-
- EXAMPLE 14
-
- Molecular Cloning and Sequencing of M. fermentans incognitus
-
- DNA was phenol extracted from sucrose-banded M. fermentans
incognitus
- derived from Sb51 cells which were first lysed by 0.5%
sodium dodecyl
- sulfate (SDS) and treated with proteinase K (200 mg/ml),
for 1 hour at
- 60.degree. C. then 3 hours at 37.degree. C. The alcohol
precipitated DNA
- was treated with RNase. An EcoRI partial digest of the
M. fermentans
- incognitus-enriched DNA was cloned into bacteriophage
lambda charon 28. The
- lambda-recombinant clones were screened by differential
plaque
- hybridization, on duplicate sets of filters, with .sup.32
P-labeled DNA
- derived from gradient banded M. incognitus versus that
of normal NIH/3T3
- cells. One clone which had specifically hybridized to
M. fermentans
- incognitus DNA probe, but not to 3T3 DNA probe was identified.
The insert
- of the positive phage clone was recloned into the EcoRI
site of Bluescript
- KS (M13) vector (Strategene). Two cloned probes, 8.6
kilobase (psb-8.6) and
- 2.2 kilobase (psb-2.2) were obtained. The specificity
of probes psb-8.6 and
- psb-2.2 was further verified by Southern blot analysis
of DNA isolated from
- M. fermentans incognitus and Sb51 cells versus normal
NIH/3T3 cells. To
- obtain sequence information, single-standed DNA of clone
psb-2.2 was
- prepared by infection of the cells with helper phage
(Bluescript
- instruction manual, Stragegene). About 200 base sequences
starting from the
- EcoRI site at one end of the insert fragment of psb-2.2
were obtained,
- using a dideoxynucleotide sequencing method. The base
sequence is set forth
- in SEQ ID NO:2.
-
-
- EXAMPLE 15
-
- Southern Blot Hybridization of M. fermentans incognitus
-
- Restriction endonuclease cleavage of M. fermentans incognitus
or cellular
- DNA was carried out with a 10-fold excess of enzymes
under the conditions
- recommended by the manufacturer (BRL).
- The enzyme digests of DNA were subjected to gel electrophoresis
in 1%
- agarose and transferred onto nitrocellulose membranes
by the Southern blot
- method. Each filter was prehybridized at 42.degree. C.
for at least 4 hours
- in 50% formamide, 5x SSC (standard saline citrate), 0.2%
SDS, 20 mM
- Tris-HCl (pH 7.5), 2 mM EDTA, 5x Denhart's solution,
and 350 microgram/ml
- denatured salmon sperm DNA. Each filter was then hybridized
with 10.sup.7
- cpm of .sup.32 P-labeled probe (specific activity after
hybridization, the
- blots were washed at 60.degree. C. in 2x SSC, 0.5% SDS
for 90 minutes with
- three changes and then wrapped in sheets of saran wrap
and exposed to Kodak
- XAR film at -70.degree. C. with intensifying screens
for 2-20 hours
- depending upon the intensity of the hybridized signals.
For the reuse of
- the membrane, the filters were boiled in 0.1x SSC, 0.1%
SDS for 10 minutes
- to remove the previous M. fermentans incognitus probe
after
- autoradiographic exposure, and rehybridized with .sup.32
P-labeled insert
- fragment of psb-8.6 as previously described.
- Use of the filters and results of such use are presented
in Example 19 below.
-
-
- EXAMPLE 16
-
- Analysis of Taq DNA Polymerase-Catalyzed PCR Amplification
Products
-
- The amplification of selective DNA sequences by Taq DNA
polymerase chain
- reaction is known (U.S. Pat. No. 4,683,202). Briefly,
each 100 microliter
- reaction mixture contained 1 microgram of human tissue
DNA in 50 mM KCl, 10
- mM Tris-HCl (pH 8.3), 1.5 mM MgCl.sub.2, each primer
(RS47 (SEQ ID NO:13)
- and RS49 (SEQ ID NO:14)) at 1 microM, each dNTP at 200
microM, gelatin at
- 100 micrograms/ml, and 2 units of Taq DNA polymerase.
The mixtures were
- heated at 94.degree. C. for 2 minutes before the addition
of DNA
- polymerase. The samples were overlaid with 50 microliters
of mineral oil
- and subjected to 35 cycles of selective DNA amplification.
The thermal
- cycle was manually conducted in three separate water
baths as follows: 1
- minute at 52.degree. C., 1 minute at 72.degree. C., and
30 seconds at
- 94.degree. C. After the amplification, the reaction was
stopped by addition
- of EDTA (final concentration, 20 mM). Ten microliter
aliquots from each
- sample product were removed and electrophoretically fractioned
in a 6%
- polyacrylamide gel. The fractionated DNA was electroblotted
onto a Zeta
- membrane (Bio/Rad) at 40 volts for 90 minutes, followed
by denaturation and
- fixation in 400 mM NaOH, 2 mM EDTA for 10 minutes at
room temperature. The
- Zeta membrane filter was rinsed three times with 2x SSC
in 20 mM Tris-HCl
- (pH 7.5), and air dried for 10 minutes. Prehybridization
of the blots was
- carried out as previously described except the solution
contained 4x SSC
- and 1% SDS. A 22-base synthetic oligonucleotide probe
(RS48 (SEQ ID NO:1)),
- was 5' end-labeled with .sup.32 P and hybridized to the
filter at
- 30.degree. C. for 16 hours in the prehybridization solution
containing 30%
- formamide. The blots were washed at 34.degree. C. in
2x SSC, 0.5% SDS for
- 45 minutes with three changes and at 37.degree. C. for
an additional 2
- minutes.
- Use of the blots and the results of such use are presented
below in Example
- 19.
- The preferred PCR assay utilizes primers RW004 and RW005
and probe RW006 as
- described in
- Example 5.
-
-
- EXAMPLE 17
-
- DNA and Antigen Dot-Blot Analysis of M. fermentans incognitus
-
- After 11 cycles of cell-free M. fermentans incognitus
transmission, the
- control and M. fermentans incognitus infected NIH/3T3
cells of Example 13
- were subjected to M. fermentans incognitus isolation
(see Example 7,
- above). Ten microliter and/or twenty microliter samples
from each fraction
- of the isopycnic sucrose gradient were first diluted
to 400 microliters
- with PBS and then dot-blotted onto nitrocellulose paper
under vacuum. The
- dot was blocked with 5% non-fat milk and reacted with
pre-immunized or
- post-immunized rabbit antiserum (1:400 in PBS) at 37.degree.
C. for 3
- hours. The blot was then developed with alkaline phosphatase
conjugated
- goat anti-rabbit IgG (1:5000, in PBS) at 37.degree. C.
for 1.5 hours,
- followed by the addition of the substrates Nitro Blue
Tetrazolium (50 mg/ml
- in 70% dimethylformide) plus 5-Bromo-4 Chloro Indolyl
phosphate (50 mg/ml)
- (Promega; Madison, Wis.). Between each of the above steps,
the blots were
- washed five times with PBS and Tween 20 (1%), five minutes
each wash. For
- homologous DNA detection, the dotted blots were alkaline
treated,
- neutralized and probed with .sup.32 P-labeled nick-translated
psb51-8.6 or
- psb51-2.2 probes as previously described in Example 14.
Use of the blots
- and the results of such use are presented below in Example
19.
-
-
- EXAMPLE 18
-
- Immunohistochemistry for Detecting M. fermentans incognitus
Antigens in
- Infected Tissues
-
- Deparaffinized sections were incubated with 10% Bovine
serum albumin (Sigma
- Chemical Co.) in Tris-buffered saline (TBS, 0.05M Tris,
pH. 7.4 saline) for
- 39 minutes, rinsed briefly with TBS, and covered with
rabbit antisera from
- Example 12 (1:100 dilution). Slides were refrigerated
overnight. After
- returning to room temperature, the slides were rinsed
with 1% albumin in
- TBS. Slides were then covered with secondary antisera.
Biotin-labelled
- horse anti-rabbit immunoglobulin (Vector Lab., Burlingame,
Calif.) was
- added at a 1:200 dilution as the secondary antisera,
followed by the avidin
- biotinylated peroxidase complex (ABC) reagent (Vector
Lab., Burlingame,
- Calif.). Each incubation step was conducted for 30 minutes
with three TBS
- washes between steps. The color reaction was developed
in Diaminobenzidine
- and H.sub.2 O.sub.2 substrate and counterstained with
hematoxylin.
-
- Rabbit antiserum which reacted specifically with M. fermentans
- incognitus-Sb51 was used to stain formalin-fixed paraffin
embedded lymph
- node and brain tissues of patients with AIDS. In the
immunohistochemical
- study, reticuloendothelial cells or macrophages in the
subcapsular sinus of
- a lymph node (Table 4, patient #1) were most often stained
positively (FIG.
- 13). Brain from the autopsy of a patient with central
nervous system
- symptoms and histopathologic evidence of subacute encephalitis
without
- known etiology, contained many positively stained degenerating
cells in
- lesions with diffuse infiltration of mononuclear lymphohistiocytes.
- Positive immunochemical reactivity was located in both
nuclei and cytoplasm
- of swollen and disrupted cells. More peculiarly, brains
from the autopsy of
- three other patients with CNS symptoms, but without histopathological
- evidence of encephalitis, also had numerous positively
stained
- inclusion-like spherical structures (FIG. 14). The structures,
most likely
- originating from neuroglial cells with unique pathological
changes, were
- inconspicuous in routine hematoxylin and eosin stained
sections.
-
- DNA from two of the three brains were available for PCR
study and had
- positive M. fermentans incognitus DNA information after
selective gene
- amplification (Table 4, patient #2 and #3). The positively
stained
- structures were more common in periventricular and perivascular
areas.
- Normal rabbit serum (Gibco Co.) and the rabbit serum
obtained before
- immunization with M. fermentans incognitus did not stain
these brains.
- Furthermore, the immunochemical reactivity of the rabbit
antiserum with
- either Sb51 cells or purified M. fermentans incognitus,
but not with normal
- NIH/3T3 cells or spontaneously transformed NIH/3T3 cells.
Eleven autopsy
- brain tissues obtained from non-AIDS patient were used
as controls. Brains
- from autopsies of patients with fatal rickettsial infection,
bacterial
- sepsis, disseminated mycobacteriosis and CNS metastatic
disease served as
- controls. No positive reaction was observed in these
control non-AIDS
- tissues.
-
-
- TABLE 4
- ______________________________________
- Clinico-Pathological Profiles of Patients
- with AIDS and Analysis of Specific DNA
- Amplification
- Lane Results of
- Clinical and/or
- Tissue Position
- DNA Ampli-
- Post Mortem DNA for in fication
- Subject
- Diagnosis PCR FIG. 14
- Analysis
- ______________________________________
- 1. 32 y.o.w. male
- 1) Spleen A-1 +++
- homosexual, 2) LN A-2 +
- PCP candida 3) Liver A-3 ++
- esophagitis 4) Brain A-4 -
- and cerebral
- toxoplasmosis
- 2. 47 y.o.w. male
- 1) Brain A-5 +++
- homosexual, 2) Liver A-6 +
- KS, and CMV
- infection,
- CNS syndrome
- 3. 24 y.o.w. male
- 1) Brain B-1 +++
- PCP, KS, and
- CNS syndrome
- 4. 37 y.o.w. male
- 1) Spleen B-2 +++
- homosexual,
- PCP, and CMV
- infection and
- KS
- 5. 45 y.o.w. male
- 1) KS B-3 ++
- homosexual,
- KS, CMV and
- PCP infection
- 6. 28 y.o.w. male
- 1) PBMC B-4 -
- homosexual
- with KS without
- OI
- 7. 43 y.o.w. male
- 1) PBMC B-5 +
- homosexual
- with KS without
- OI
- 8. 26 y.o.b. male
- 1) LN Not -
- homosexual shown
- with KS without
- OI
- 9. 24 y.o.w. male
- 1) Spleen Not -
- homosexual shown
- with KS and
- myocarditis
- 10. 31 y.o.w. male
- 1) Spleen Not +
- homosexual shown
- with KS, PCP,
- CMV and MAI
- infections
- 11. Diffuse 1) Spleen A-7 -
- histiocytic
- malignant
- lymphoma
- 12. Renal cell 1) Liver A-8 -
- carcinoma 2) Brain A-9 -
- 13. Chronic active
- 1) PBMC B-6 -
- hepatitis B.
- 14. Metastic Ewing
- 1) Ewing B-7 -
- sarcoma in sarcoma
- lung and liver.
- 15. Normal delivery
- 1) Placenta
- B-8 -
- placenta.
- ______________________________________
- Labels +++, ++, + and - denote highlevel, intermediate
level, low level
- and negative, respectively, for the relative intensities
of the diagnosti
- band observed in the autoradiograms in FIG. 15.
- OI Opportunistic Infection
- MAI Mycobacterium AviumIntracellular
- KS Kaposi's sarcoma
- LN Lymph node
- PBMC Peripheral Blood Mononuclear Cells
- PCR Polymerasechain reaction
-
-
- EXAMPLE 19
-
- DNA Probes for the Direct Detection of M. fermentans
incognitus DNA in
- Infected Tissues
-
- DNA was extracted from the fractions of Example 6 and
digested with EcoRI
- enzyme. Two molecular clones carrying 8.6 kb and 2.2
kb inserts, designated
- psb-8.6 and psb-2.2, were obtained. When used as probes,
these clones
- specifically hybridized to DNA of Sb51 cells (lanes 1,
2) but not to that
- of parental NIH/3T3 cells (lanes 3, 4) (FIG. 15). These
cloned probes were
- used to assay infectivity of M. fermentans incognitus
in cell cultures. The
- gradient banded M. fermentans incognitus from Sb51 cells
infected normal
- NIH/3T3 cells after being filtered through at 0.22 micron
filter. The
- psb-8.6 probe specifically hybridized to DNA of NIH/3T3
cells which were
- harvested after each round of cell-free M. fermentans
incognitus
- transmission (FIG. 16). Blotted filter containing 10
ug EcoRI digested DNA
- from cells of sb.sub.51 (lane 1), original, normal NIH/3T3
cells (lane 2),
- 7th cycle cell-free VLIA transmission control NIH/3T3
cells (lane 3), 11th
- cycle cell-free VLIA transmission control NIH/3T3 cells
(lane 4), and 3rd
- cycle (lane 5), 5th cycle (lane 6), 7th cycle (lane 7),
9th cycle (lane 8),
- and 11th cycle (lane 9) of cell-free VLIA transmission
in NIH/3T3 cells.
- Lane 10 contained DNA of partially purified VLIA. The
blot was probed with
- p.sup.32 labeled psb.sub.51 -8.6. Similarly, the psb-2.2
probe also
- specifically hybridized to DNA from M. fermentans incognitus
infected
- NIH/3T3 cells in each cycle of passage, but not from
control NIH/3T3 cells.
-
- The .sup.32 P-labeled psb-8.6 probe was also used for
detection of M.
- fermentans incognitus in isopycnic sucrose gradients
which were originally
- designed to band M. fermentans incognitus from Sb51 cells.
M. fermentans
- incognitus isolated after 11 cycles of cell-free passage
in NIH/3T3 cells
- had similar physical properties and was concentrated
in the fractions of
- density between 1.17 and 1.20 (gm/ml) (FIG. 17A). The
parallel control
- NIH/3T3 cultures following 11 cycles of cell-free transmission
did not
- contain M. fermentans incognitus. Immunochemical staining
by rabbit
- antiserum raised against M. fermentans incognitus originally
isolated from
- Sb51 cells also revealed that M. fermentans incognitus
was localized in
- these fractions (FIG. 18). FIG. 18A was stained using
preimmunized rabbit
- serum and FIG. 18B was stained with post-VLIA immunizations
rabbit
- antisera. Gel electrophoretic analysis of the end-labeled
EcoRI or HindIII
- digests of the gradient-banded M. fermentans incognitus
indicated a minimum
- molecular weight estimate for M. fermentans incognitus
of greater than
- 150,000 bp.
-
- To determine whether there was any significant homology
of M. fermentans
- incognitus to large human DNA viral agents, Southern
blot hybridizations
- were performed with each filter containing the restriction
enzyme-treated
- DNA from purified M. fermentans incognitus, NIH/3T3,
and one of the
- following viral genomic DNA: HSV-2, VZV, EBV, CMV, HBLV,
vaccinia pox virus
- and mouse CMV virus. Each filter was hybridized to .sup.32
P-labeled
- corresponding viral DNA probe, then washed and analyzed
by autoradiography.
- The incorporated viral probe was subsequently removed
by boiling the
- filters before rehybridization with .sup.32 P-labeled
insert fragment of
- psb-8.6. No cross-hybridizations of M. fermentans incognitus
probe psb-8.6
- occurred to any of the human herpesviruses, vaccinia
pox virus or mouse CMV
- (FIG. 19). Southern blot hybridization comparing VLIA
DNA to DNA from known
- human herpesviruses, vaccinia virus, MCMV, and HVS. Blotted
filters
- contained DNA of VLIA (A-H, lanes 1, 2), normal NIH/3T3
(A-H, lanes 3, 4),
- HSV-2 (A, lanes 5, 6) VZV (B, lanes 5, 6), EBV (C, lanes
5, 6), CMV (D,
- lanes 5, 6), HBLV (E, lanes 5, 6), vaccinia virus (F,
lanes 5, 6), MCMV (G,
- lanes 5, 6), and HVS pT (H, lane 5). DNA in lanes 1,
3, and 5 were digested
- by Eco RI: DNA in lanes 2, 4, and 6 were digested by
Bam HI. HVS pT 7.4 (H,
- lane 5) was digested with Taq I. The p.sup.32 labeled
probes for set I were
- HSV-1 pHSV-106 (A), VZV pEco A (B), EBV pBam W (C), CMV
pCMH-35 (D), HBLV
- pZVH-70 (E), vaccinia pEH-1 (F), MCMV pAMB-25 (G), and
HVS pT 7.4 (H). Each
- blot (A-H) of set I was boiled to remove incorporated
viral probe and then
- reprobed with p.sup.32 labeled insert fragment of psb-8.6
(set II).
-
- Conversely, while they hybridized to the homologous genomic
DNA, one of the
- other viral probes hybridized to the lanes containing
M. fermentans
- incognitus DNA digested with either EcoRI or BamHI. pHSV-106
originated
- from HSV type I hybridized to the genomic DNA of HSV
type II, but not to
- the M. fermentans incognitus DNA.
-
- A viral probe of 7.4 kb DNA (pT 7.4) from Herpesvirus
saimiri (HVS) of
- squirrel monkeys was also examined. The viral probe did
not hybridize to M.
- fermentans incognitus DNA. In some of the rehybridized
filters, very weak
- bands resulting from incomplete removal of the previously
hybridized viral
- probes could be noted. The weak signals served as useful
reference points
- for the newly appearing bands obtained after rehybridization
with psb-8.6
- probe.
-
- To investigate M. fermentans incognitus infection in
humans, the recently
- developed, a sensitive method of selective DNA amplification,
polymerase
- chain reaction (PCR) (U.S. Pat. No. 4,683,202) was used.
(As discussed
- above in Example 5, it is preferred to use RW004 (SEQ
ID NO:15) and RW005
- (SEQ ID NO:16) as primers because a more sensitive assay
is possible with
- these primers.) One end of the M. fermentans incognitus
DNA in the psb-2.2
- bluescript clone was sequenced. Primer pairs of synthetic
oligonucleotides,
- designated as RS47 (SEQ ID NO:13) and RS49 (SEQ ID NO:14),
with M.
- fermentans incognitus-specific sequences and Taq DNA
polymerase were used
- for 35 reaction cycles of M. fermentans incognitus specific
DNA
- amplification. The positions of RS47 (SEQ ID NO:13) and
the complementary
- sequences of RS49 (SEQ ID NO:14) span the first 160 nucleotides
of psb-2.2
- M. fermentans incognitus DNA (SEQ ID NO:2). The amplified
DNA carrying M.
- fermentans incognitus-specific genetic information revealed
positive
- signals, when probed with .sup.32 P end-labeled synthetic
oligonucleotides
- RS48 representing a segment of the intervening sequences
between RS47 (SEQ
- ID NO: 13) and RS49 (SEQ ID NO:14).
-
- Ten patients with AIDS have been examined and were seropositive
for HIV and
- had either typical opportunistic infections such as pneumocystic
carinii
- pneumonia (PCP), toxoplasmosis, CMV infection or Kaposi's
sarcoma (Table 4,
- subjects #1 to #10). Analysis of the amplified DNA products
revealed that a
- diagnostic 160 bp DNA fragment and a slower migrating
fragment(s)
- associated with a positive homologous signal, were identified
in samples
- derived from seven of the ten AIDS patients tested. Representative
results
- of nine positive samples and two negative samples obtained
from seven AIDS
- patients are shown in FIG. 20. No positive signal could
be detected in any
- of the six DNA samples derived from five control non-AIDS
subjects (Table
- 4, subjects #11 to #15). As summarized in Table 4, patient
#1 had M.
- fermentans incognitus genetic material in the lymph node,
liver and spleen
- but not in the brain. However, both patients #2 and #3
had positive M.
- fermentans incognitus-specific DNA products in the brain
samples.
-
-
- EXAMPLE 20
-
- Vaccine Containing Cells Infected by M. fermentans incognitus
-
- Sixteen chimpanzees are divided into four groups. Group
A is inoculated
- intravenously with 1 ml of the novel M. fermentans incognitus.
Group B is
- inoculated with 1 ml of fluid containing 10.sup.6 M.
fermentans
- incognitus-infected NIH/3T3 cells. Group C is inoculated
with 1 ml of fluid
- containing 10.sup.6 inactivated M. fermentans incognitus-infected
NIH/3T3
- cells, and Group D is the control group and did not receive
an inoculation.
-
- All chimpanzees in Groups A and B developed symptoms
of AIDS. However, none
- of the chimpanzees in Groups C and D developed the symptoms
of AIDS. The
- chimpanzees of Group C are rendered immune to subsequent
challenge of
- intravenous inoculation with 1 ml of M. fermentans incognitus
or 1 ml
- containing 10.sup.6 M. fermentans incognitus-infected
NIH/3T3 cells.
-
-
- EXAMPLE 21
-
- M. fermentans incognitus Identified In Non-AIDS Patients
-
- Six patients from six different geographic areas who
presented with acute
- flu-like ilnesses were studied. The patients developed
persistent fevers,
- lymphadenopathy or diarrhea, pneumonia, and/or heart,
liver, or adrenal
- failure. They all died in 1-7 weeks.
-
- These patients had no serological evidence of HIV infection
and could not
- be classified as AIDS patients according to CDC criteria.
The clinical
- signs as well as laboratory and pathological studies
of these patients
- suggested an active infectious process, although no etiological
agent was
- found despite extensive infectious disease work-ups during
their
- hospitalization.
-
- Post-mortem examinations showed histopathological lesions
of fulminant
- necrosis involving the lymph nodes, spleen, lungs, liver,
adrenal glands,
- heart, and/or brain. No viral inclusion cells, bacteria,
fungi, or
- parasites could be identified in these tissues using
special tissue stains.
- However, the use of rabbit antiserum and the monoclonal
antibodies raised
- against M. fermentans incognitus (Example 8), the pathogen
shown to cause
- fatal systemic infection in primates (Example 10), revealed
M. fermentans
- incognitus antigens in these necrotizing lesions. In
situ hybridization
- using a .sup.35 S labeled M. fermentans incognitus-specific
DNA probe
- (Example 18) also detected M. fermentans incognitus genetic
material in the
- areas of necrosis.
-
- Furthermore, M. fermentans incognitus particles were
identified
- ultrastructurally in these histopathological lesions.
M. fermentans
- incognitus was associated with the systemic necrotizing
lesions in these
- previously healthly non-AIDS patients with an acute fatal
disease.
-
- Typical areas of necrosis due to the M. fermentans incognitus
infection of
- these patients are shown in FIG. 21. Most of the tissues
which had massive
- necrosis showed only minimal lymphocytic or histiocytic
response and few
- neutrophils (FIGS. 21A, B and C). FIG. 21A is a photomicrograph
of splenic
- tissue (x 30.5). FIG. 21B shows the peripheral margin
of necrosis of 21A (x
- 153). FIG. 21C is a photomicrograph of lymph node tissue
(x 15.25).
- Occasionally, a chronic or acute inflammatory reaction
could be identified
- in the areas of necrosis (FIG. 21D). FIG. 21D is a photomicrograph
of
- adrenal gland tissue (x 153).
-
- Representative samples of the immunostained tissues of
these patients are
- shown in FIGS. 22A-D. FIG. 22A is a photomicrograph of
spleen tissue (x
- 80). FIG. 22B is a higher magnification of the margin
of necrosis of 22A (x
- 353). FIG. 22C is a photomicrograph of lymph node tissue
(x 257). FIG. 22D
- is a higher magnification of cells with positive cytoplasmic
staining of
- 22C (x 706). FIG. 22E is a photomicrograph of hemorrhagic
necrosis in
- adrenal gland tissue (x 706). The areas which displayed
the highest
- concentration of M. fermentans incognitus related antigens
were often at
- the margin of necrosis.
-
- However, the necrotic center and peripheral unaffected
areas had relatively
- low reactivity. Most of the positively stained cells
were identified as
- lymphocytes or histiocytes in the lymph nodes and spleen,
or reactive
- mononuclear cells in the liver, lungs, adrenal glands
and heart.
-
- Immunostaining of control tissues with necrotizing lesions
from patients
- with cat scratch disease, Hodgkin's disease, malignant
lymphoma,
- cryptococcal fungal infections and hemorrhagic splenic
tissues of Hairy
- cell leukemia did not display a positive reaction. Serum
obtained from the
- same rabbit before immunizaiton with M. fermentans incognitus
antigens also
- failed to display a positive immunoreaction in the necrotizing
lesions of
- the six patients.
-
- Using a .sup.35 S radiolabeled psb-2.2 M. fermentans
incognitus DNA probe
- (Example 18), strong labeling of clusters of cells at
the margins of
- necrosis of the affected tissues was observed. The affected
tissues tested
- were formalin-fixed, paraffin-embedded spleen, lung,
lymph node, adrenal
- gland liver and bone marrow. The intensity of the labeling,
or the number
- of grains localized in the cells at the margin of necrosis
was well above
- the level present at either the necrosis (FIGS. 23A and
B). However, there
- were also clusters of apparently viable cells in the
necrosis which were
- also strongly labeled (FIG. 23C). FIG. 23A shows strong
labeling of cells
- at the peripheral zone of necrosis (x 76.5). FIG. 23B
is a higher
- magnification of 23A (x 422). FIG. 23C shows the occasional
positive
- labeling in an area of diffuse necrosis in the spleen
(x 150). The inset of
- 23C is a higher magnification (x 422).
-
- Formalin-fixed, paraffin-embedded liver and spleen tissues
from a patient
- with pancreatic carcinoma were used as negative controls,
and showed no
- labeling above background levels. A control probe of
.sup.35 S labeled
- cloning vector DNA, not containing psb-2.2 M. fermentans
incognitus DNA did
- not label any of the tested tissues (FIG. 23D). FIG.
23D is the same area
- of FIG. 23C in the consecutive tissue section, hybridized
with .sup.35 S
- labeled cloning vector DNA not containing psb-2.2 M.
fermentans incognitus
- DNA (x 150) (i.e., control for 23C).
-
- Areas of the necrotizing lesions which immunostained
most positively for M.
- fermentans incognitus specific antigens were examined
by electromicroscopy.
- Particles with characteristic ultrastructural features
of M. fermentans
- incognitus were directly identified in all the lesions.
These particles in
- the areas of necrosis, morphologically resembled M. fermentans
incognitus
- previously identified in Sb51 cells (Example 4) and in
the tissues of
- experimentally inoculated monkeys (Example 10). The particles
were
- heterogeneous in size and shape, with most particles
being spherical and
- about 140 to 280 nm in diameter. At the margin of necrosis,
the M.
- fermentans incognitus particles were located in the cytoplasm
of cells with
- apparently no cytopathic changes, or in fragments of
cytoplasm from
- completely disrupted cells (FIG. 24). FIG. 24 shows electron
mircographs of
- tissues derived from areas highly positive for M. fermentans
- incognitus-specific antigens. FIG. 24A is an electron
micrograph at a
- margin of necrosis in adrenal gland tissues (Bar=1,000
nm). FIG. 24A.sub.2
- is a higher magnification of 24A (Bar=100 nm). FIGS.
24B.sub.1, and B.sub.2
- are electron micrographs of the peripheral zone of necrosis
in lymph node
- tissue (Bar=1,000 nm). FIG. 24B.sub.3 is a higher magnification
of
- 24B.sub.2 (Bar=100 nm).
-
- Table 5, below, summarizes the profiles and histopathological
findings for
- each of the six patients.
-
-
- TABLE 5
- __________________________________________________________________________
- Summary of Patient's Profiles and Histopathological
Findings
- Tissue with necrotic
- Duration
- lesions identified
- Personal
- Salient clinical of illness by biopsy or at
Patient
- Profiles presentation (weeks) autopsy
- __________________________________________________________________________
- 1 29-year old
- arthralgia, myalgia, conjunc-
- 4.5 spleen, lung
- black man
- tivitis, persistent fever,
- hypercalcemia, liver failure
- (late), ARDs* (late)
- 2 33-year old
- persistent fever, diarrhea,
- 7 lymph nodes,
liver,
- white woman
- generalized lymphadenopathy,
- spleen, kidneys
- abnormal liver functions,
- seizure (late)
- 3 40-year old
- arthralgia, myalgia, sore
- 3.5 adrenal glands
- white man
- throat, chest pain, persis-
- (bilateral),
heart,
- tent fever, malaise, diarrhea,
- brain
- finger numbness, comatose
- (late)
- 4 31-year old
- vomiting and diarrhea, tremor,
- 1.5 liver, spleen
- black woman
- fever, epigastric and chest
- pain, abnormal liver functions,
- headache
- 5 23-year old
- Watery diarrhea, vomiting,
- 3 liver, heart
- white man
- jaundice, arthralgia, myalgia
- 6 33-year old
- fever, malaise, nausea and
- 1 spleen, liver
- black man
- vomiting, myalgia and weakness,
- liver failure and jaundice,
- confusion and hallucinations
- (late)
- __________________________________________________________________________
- *ARDS Adult Respiratory Distress Syndrome
-
-
- EXAMPLE 22
-
- Biochemical Properties and Characteristics of M. fermentans
incognitus
-
- In order to identify biochemical properties and characteristics
of M.
- fermentans incognitus, a variety of analyses were performed
on this
- pathogen. The analyses of biochemical properties, antigenic
specificity,
- DNA homology and restriction pattern analysis show that
M. fermentans
- incognitus is distinct from all other know species of
human mycoplasma, but
- appears to be biologically, sereologically and molecular
incognitus, a
- rarely isolated human mycoplasma genetically most closely
related to M.
- fermentans, a rarely isolated human mycoplasma.
-
- M. fermentans incognitus from culture supernatant of
Sb51 cells (Example 4)
- was cultured in cell-free conditions using a modified
SP-4 medium. SP-4
- broth was prepared according to previously described
procedures (Whitcomb,
- R. F., Methods in Mycoplasmology, Vol. I, Academic Press,
Inc. pp. 147-158
- (1983) and Tully, J. G. et al., Science 195, 892 (1977)),
and then
- supplemented with 20% heat inactivated fetal bovine serum
(FVS) (M.A.
- Bioproducts Cat. #14-901B, Lot No. 8M0320 for hybridoma).
Modified SP-4
- broth medium was further supplemented with 0.15 mg/ml
niacin (nicotinic
- acid, Sigma), 0.15 mg/ml riboflavin (Sigma) 0.15 mg/ml
L-arginine and 0.01
- mg/ml nicotinamide adenine dinucleotide (NADH, Pharmacia).
Modified SP-4
- agar medium containing 1% Noble agar (Gibco) was dispensed
into sterile
- plastic petri-plates (Falcon).
-
- The cell debris from the Sb51 cells was first removed
from 5 day-old
- culture supernatant by centrifugation at 1,500 rpm for
15 minutes.
- Thesupernatant was then pelleted in Sorvall superspeed
centrifugation
- 10,000 rpm for 20 minutes. The particles pelleted from
50 ml of culture
- supernatant were resuspended in 1 ml of modified SP-4
medium and used as
- inoculum. The M. fermentans incognitus-containing suspension
was 1:10 fold
- serially diluted with SP-4 medium and then inoculated
(0.2 ml) into
- modified SP-4 broth culture medium (2 ml).
-
- Culture incubation and observation
-
- All broth cultures and agar media plates were either
incubated at
- 37.degree. C. or 30.degree. C. in anaerobic Gas Pak jars
(BBL, Microbiology
- Systems, Cockeyville, Md.), candle jars or in a regular
incubator. The
- broth media were examined daily for three weeks. Broth
cultures were
- observed macroscopically against a white background to
facilitate detection
- of color changes. Positive broth cultures were confirmed
by subculturing
- 0.1 ml volumes to fresh modified SP-4 both and agar plates
as soon as any
- color change was detected.
-
- The surface of the agar plates was scanned with the use
of a low-power
- objective (X4) from a standard light microscope or an
inverted microscope.
- Positive cultures were identified by characteristic colony
morphology.
-
- For the studies of antigenic and DNA analysis, M. hyorhinis
9ATCC #17981),
- M. orale (ATCC #23714), M. pneumonia (ATC #15531), M.
hominis (ATCC
- #15488), M. genitalium (ATCC #33530), M. salvarium (ATCC
#23064), M.
- fermentans incognitus and Acholeolasma laidlawii (ATCC
#23206) strains were
- cultured in modified SP-4 broth. U. urealyticum (ATCC
#27618) was cultured
- in modified SP-4 broth supplemented with 0.03% urea.
-
- The broth cultures appeared slightly turbid and an acidic
shift in pH
- occurred after 10 to 14 days of incubation either at
30.degree. C. or
- 37.degree. C. Cells grew slightly better in a candle
jar than in aerobic
- conditions; observation of a pH shift usually occurred
about one day
- earlier.
-
- M. fermentans incognitus could be filtered through a
220 nm membrane filter
- and continued to grow in the broth filtrate. The cells
grown in the
- modified SP-4 broth were examined by electron microscopy
after either
- ultrathin sectioning or direct negative staining. Clusters
of cell
- wall-free microorganisms which were bound by a single
triple layered
- membrane, showed typical plemorphic morphology of Mollicutes.
-
- Most of the particles were spherical, but filamentous
forms with occasional
- branching configuration, were also observed (FIG. 1A).
In general, the
- average size of spherical M. fermentans incognitus particles
in the broth
- cultures appeared to be much smaller than that of M.
fermentans (180 nm
- versus 460 nm).
-
- M. fermentans incognitus could also produce colonies
on 1% Noble agar
- plates prepared from modified SP-4 media. Compared with
some other human
- mycoplasmas, M. fermentans incognitus grew rather slowly
and formed only
- small colonies (FIG. 1C). For comparison, colonies with
a regular size and
- sharp edge formed by M. fermentans incognitus growing
in a parallel
- modified SP-4 medium agar plate after a shorter incubation
period are shown
- in FIG. 1D. The small colonies of M. fermentans incognitus
became
- microscopically visible after 10 to 14 days of incubation.
Most of the
- colonies were somewhat diffuse and irregular, and much
of their growth
- occurred within the agar. However, under an inverted
phase microscope, the
- small central area of the colony was found to grow even
deeper into the
- agar and exhibited the appearance of a "fried egg"
(FIG. 1C).
-
- A single typical colony of M. fermentans incognitus was
picked three times
- from consecutive agar plates. The cloned agent was then
continuously grown
- and passed in the broth of modified SP-4 medium. There
was no evidence of
- cell wall growth or conversion into a bacterium, when
M. fermentans
- incognitus was cultured and passed in an antibiotic-free
medium.
-
- In order to verify the definite relationship between
M. fermentans
- incognitus and what was previously identified as VLIA
from Sb51 cells
- (prior patent application Ser. No. 265,920, filed Nov.
2, 1988), DNA from
- this cloned M. fermentans incognitus was isolated and
compared with that of
- Sb51 cells containing VLIA. The DNAs were first digested
with EcoRI,
- HindIII and PstI restriction enzymes. In the analysis
of a Southern blot
- probed with either psb-8.6 or psb-2.2, DNA of M. fermentans
incognitus
- grown in a cell free condition using modified SP-4 medium
was identical to
- DNA of VLIA in Sb51 cells (FIG. 25). This tertially cloned
M. fermentans
- incognitus was later used for all the following assays
in this study.
-
- FIG. 25 shows analysis and comparison of DNA restriction
patterns of VLIA
- and M. fermentans incognitus. Blot (A) and blot (B) were
probed with
- .sup.32 P nick translated inserts of psb-8.6 and psb-2.2,
respectively.
- Each lane in the gel contained 1 microgram of DNA from
sb51 cells infected
- with VLIA (lanes 1,2,3) and control NIH/3T3 cells (lanes
4,5,6) or 1
- nanogram of DNA from M. fermentans incognitus cultured
in modified SP-4
- broth (lanes 7,8,9). DNA was predigested with restriction
enzymes EcoRI
- (lanes 1,4,7) HindIII (lanes 2,5,8) and PstI (lanes 3,6,9).
Arrows
- indicated the positions of standard size maker 23, 9.4,
6.7, 4.4, 2.3, and
- 2.0 kbp, respectively.
-
- Biochemical characterization
-
- The tests of glucose breakdown by oxidation or fermentation,
and hydrolysis
- of arginine or urea were performed according to standard
bacteriological
- techniques for the characterization of mycoplasma species
(Alvotto, B. B.
- et al., Intl. J. Systematic Bacteriology 20, 35 (1970)).
Specifically,
- glucose, arginine and urea media were prepared by adding
10 ml of 10% (w/v)
- test substrate and 1 ml of 0.5% (w/v) phenol red to 74
ml of modified SP-4
- broth without glucose. Each medium was adjusted using
5N HCl or 4N NaOH to
- the following initial pH values: glucose medium, 7.6;
arginine medium, 7.0;
- and urea medium, 7.0. Each broth medium was filtered
through a 0.22
- micrometer filter and dispensed in 5 ml amounts into
screw-capped tubes.
-
- All inoculated cultures were incubated at 37.degree.
C. Anaerobic cultures
- were kept in Gas Pak jars (Gibco) and candle jars. Tests
were read daily. A
- drop of 0.5 pH unit or more in the glucose tube compared
with the
- appropriate substrate control tube constituted a positive
reaction; a rise
- of 0.5 pH unit or more in the arginine or urea tubes
compared with the
- appropriate substrate control tubes constituted a positive
test. The pH
- values were read by comparison with a set of standards
ranging from pH 5.6
- to 8.4. Positive and negative test control organisms
were:
-
- A) Glucose breakdown (both aerobic catabolism and fermentation)
- Positive: M. fermentans and M. hyorhinis
- Negative: M. orale
-
- B) Arginine hydrolysis:
- Positive: M. fermentans and M. orale
- Negative: M. hyorhinis
-
- C) Urea hydrolysis:
- Positive: Ureaplasma urealyticum
- Negative: M. fermentans
-
- In comparison with other known species of human mycoplasmas,
including M.
- pneumoniae and M. fermentans incognitus, M. fermentans
incognitus appeared
- to be more fastidious in cultivation and did not grow
in the conventional
- mycoplasma media (Table 5, presented at the end of this
Example). Modified
- SP-4 (with the further addition of NADH, niacin and riboflavin)
was the
- only medium able to support a continuous growth of M.
fermentans
- incognitus. Serum was a necessary supplement which could
not be replaced by
- albumin. Increased fetal bovine serum concentrations
(at least up to 10 to
- 15% of supplement) in the modified SP-4 medium produced
a growth response.
-
- M. fermentans incognitus catabolized glucose under both
aerobic and
- anaerobic conditions of cultivation (Table 6). M. fermentans
- incognitushydrolyzed arginine and produced an alkaline
shift in pH, albeit
- slower than M. fermentans incognitus. A prominent alkaline
shift in pH
- occurred after an initial brief acidic shift in the M.
fermentans
- incognitus broth culture. M. fermentans incognitus could
not hydrolyze urea
- in the bichemical assay. The usual biological characteristics
of this
- microorganism are apparently distinct from all the other
human species but
- similar to M. fermentaas, another glycolytic and arginine-metabolizing
- mycoplasma (Kenny, G. E., Manual of Clinical Microbiology,
American Society
- for Microbiology, Washington, D.C 4th Ed., pp.d 147-158)
(1985)).
-
-
- TABLE 6
- __________________________________________________________________________
- Comparison of Growth and Biochemical Properties of
Mycoplasma
- incognitus to Eight Other Mollicutes
- Species
- AL MA MHO MHY MP MO UU
MF MI
- __________________________________________________________________________
- (I) Ability of Growth in Different
- Culture Media*:
- Hayflick + + ND ND + + ND
+ -
- Brain & Heart Infusion Broth
- + ND ND ND ND ND ND
.+-.
-
-
- Mycotrim-TC + ND ND ND ND + ND
+ -
- Heart Infusion Broth
- + ND ND ND ND + ND
+ -
- Arginine Broth + + ND ND ND ND ND
+ -
- Boston Broth + + ND ND ND ND ND
+ -
- A7 Agar + + ND ND ND ND ND
+ -
- SP-4 + + ND ND ND ND ND
+ .+-.
- Modified SP-4 + + + + + + +**
+ +
- (aerobic and candle jar)
- (II)
- Biochemical Properties:
- Glucose Breakdown
- Oxidation (aerobic culture)
- ND - ND + ND - ND
+ +
- Fermentation (anaerobic
- ND - ND + ND - ND
+ +
- culture)
- Arginine Hydrolysis
- ND + ND - ND + ND
+ +
- Urea Hydrolysis ND ND ND ND ND ND +
- -
- __________________________________________________________________________
- *All the culture media were supplemented with 20%
fetal bovine serum.
- **The SP4 medium was supplemented with urea.
- AL: A. laidlawii, MA: M. arginini, MHO: M. hominis,
MHY: M. hyorhinis, MP
- M. pneumoniae, MO: M. orale, UU: U. urealyticum,
MF: M. fermentans, MI: M
- incognitus ND: Not done in this study.
-
-
- Southern blot DNA analysis
-
- Restriction endonuclease cleavage and Southern blot hybridization
using
- nick translated psb-8.6 nd psb-2.2 probes as well as
.sup.32 P end-labeled
- RS48 were described previously (Examples 13-17). A cDNA
probe of E. coli
- r-DNA (23S and 16S r-RNA, Pharmacia Cat. #27-2508-01)
was prepared with
- .sup.32 P alphadeoxyadenosine triphosphate by random
primer extension
- method (Feinberg, A. P. et al., Anal. Biochem. 132, 6
(1983)) using cloned
- Moloney murine leukemia virus reverse transcriptase (from
BRL) and random
- primer (Pharmacia) under the conditions recommended by
the manufacture of
- BRL. Two tenth micrograms of purified DNA isolated from
cultures of each
- species of mycoplasa were applied to each lane for gel
electrophoresis
- after restriction enzyme digestion.
- Molecular cloning of M. fermentans incognitus DNA
-
- DNA was phenol extracted from a pure culture of M. fermentans
incognitus
- grown in modified SP-4 medium. The alcohol precipitated
DNA was treated
- with Rnase. A HindIII digest of the M. fermentans incognitus
DNA was cloned
- into M13 mp18 Vector (Norrander, J. et ll., Gene 26,
101 (1983)). The M13
- mp18 recombinant clones were screened by plaque hybridization,
on
- nitrocellulose filters, with .sup.32 P-labeled DNA derived
from M.
- fermentans incognitus. One clone which had specifically
hybridized to M.
- fermentans incognitus DNA probe was identified. The insert
of 3.3 kilobase
- M. fermentans incognitus DNA (MI-H 3.3) was identified
in the cloned probe.
- The cloned probe MI-H 3.3 used for Southern blot DNA
analysis, had been
- radiolabeled with .sup.32 P alpha-deoxyadenosine triphosphate
by the chain
- elongation method (Lo, S-C et al., Am. J. Trop. Med.
Hyg. 41, 380 (1989)
- and Messing, J. et al., Methods of Enzymology Vol. 101,
Academic Press,
- Inc., pp-2078 (1983)) using the M13 universal sequencing
primer (17 mer,
- USBC Co.) and the Klenow fragment of DNA polymerase I
(USBC Co.).
-
- Development and isotyping of monoclonal antibodies
-
- Balb/c mice were immunized with heat inactivated (60.degree.
C. for 20
- minutes) M. fermentans incognitus in complete Freund's
adjuvant through the
- interperitioneal route. The mice were subsequently boosted
twice at
- biweekly intervals, three weeks after the initial injection,
with heat
- inactivated M. fermentans incognitus material in incomplete
Freund's
- adjuvant. Four days after the last boost, the spleen
was removed and the
- spleen cells were fused with NS1 myeloma cells using
polyethylene glycol as
- described in Galfre and Milstein (Methods of Enzymology
Vol. 73, Academic
- Press, Inc., pp. 3-46 (1981)).
-
- The fused cells were then added to 96-well microtiter
plates in
- hypoxanthine, aminopterin and thymidine supplemented
medium to eliminate
- unfused myeloma cells. Culture supernatants in each well
were then tested
- for the production of antibody by using M. fermentans
incognitus
- antigen-coated microtiter plates in an ELISA system.
-
- Selected hybridomas were cloned by the limiting dilution
assay in 96-well
- microtiter plates. Supernatants from wells demonstrating
active growth were
- re-tested for antibody activity in the ELISA system.
The specificity of the
- monoclonal antibodies was further crossed-checked by
using M. fermentans
- incognitus, Sb51 and NIH/3T3 cell antigen-coated microtiter
plates. The
- generation of ascites fluid was accomplished by injecting
ten million
- hybridoma cells into the perioneal cavity of Balb/c Nu/Nu
mice which had
- been primed with 0.5 ml of pristane, 5-7 days earlier.
Ascites were
- harvested by inserting a 20 gauge needle and withdrawing
the fluid. The
- material was clarified by centrifugation at 2500 rpm
(300x g) for 10
- minutes, and stored at -70.degree. C. Isotyping was done
using reagents
- from isotyping kit (Screentype, Boehringer Mannheim Biochemicals)
and
- Bio-Dot apparatus (Bio-Rad).
-
- Analysis of genomic DNA by restriction enzyme mapping
and comparison of
- specific sequence homology were extremely useful in comparing
different
- species of mycoplasma. Ten different species of mycoplasma,
M. orale, M.
- hyorhinis, M. pneumonia, M. arginini, M. hominis, M.
fermentans, M.
- genitalium, M. salivarium, U. urealyticum and A. laidlawii
were obtained
- from ATCC and cultured in the modified SP-4 broth medium
with or without
- specific supplement. DNA isolated from M. fermentans
incognitus and these
- mycoplasmas was analyzed on Southern blots and probed
with .sup.32 P
- labeled cloned M. fermentans incognitus DNA (psb-8.6,
psb-2.2) or synthetic
- oligonucleotide (RS48).
-
- FIG. 3 shows a comparison of DNA homology and restriction
patterns between
- M. fermentans incognitus and other human mycoplasmas.
The blots were probed
- with .sup.32 P.sub.-- translated, psb-8.6 (A), psb-2.2
(B), .sup.32 P
- end-labelled RS48 (C), .sup.32 P labeled MI-H 3.3 (D)
and .sup.32 P labeled
- cDNA probe of E. coli ribosomal RNA (E). Each lane contained
0.2 microgram
- of EcoRI enzyme pre-digested DNA from Acholeplasma laidlawii
(lane 1), M.
- arginini (lane 2), M. hominis (lane 3), M. hyorhinis
(lane 4), M.
- pneumoniae (lane 5), M. orale (lane 6), M. fermentans
incognitus (lane 7)
- and M. fermentans incognitus (lane 8). Arrows indicate
the positions of
- standard size marker 23, 9.4, 6.7, 4.4, 2.3, and 2.0
kbp, respectively.
-
- One additional molecular clone, carrying the 3.3 kilobase
insert of M.
- fermentans incognitus DNA, designated MI-H 3.3, was also
used as a probe in
- the study. Although some homology with psb-2.2 was observed
in the M. orale
- genome (FIG. 3B), no homology with RS48 (SEQ ID NO:1),
the specific DNA
- sequences occurring at one terminal end of psb-2.2, and
no homology with
- psb-8.6 or MI-H 3.3 could be identified in the M. orale
genome.
-
- However, DNA homology with psb-8.6, psb-2.2, RS48 and
MI-H 3.3 were all
- found in the M. fermentans genome (FIG. 3A, B, C, D),
but, the restriction
- patterns revealed by these probes were different between
M. fermentans and
- M. fermentans incognitus. No similar DNA homology could
be found in any
- other species of mycoplasma.
-
- There is significant homology between the ribosomal RNA
(r-RNA) genes of
- procaryotic mycoplasmas and those of Escherichia coli
bacterium (Gaobel, U.
- B. et al., Science 226, 1211 (1984)). The same blot which
had been probed
- consequently with RS48 and MI-H 3.3, was reprobed with
.sup.32 P labeled
- cDNA of E. coli r-RNA, after removing the previously
incorporated probes by
- boiling the filter. This analysis of r-RNA genes revealed
both a difference
- in numbers and size of the hybridization bands with each
different species
- of mycoplasma tested (FIG. 3E). The characteristic restriction
enzyme
- mappings of r-RMA genes in these Mollicutes enable the
identification of
- related species. The EcoRI restriction pattern of r-RNA
genes of M.
- fermentans incognitus and M. fermentans appeared to be
identical (FIG. 3E)
- and was different from any other mycoplasma tested.
-
- Antigenic analysis using polyclonal and monoclonal antibodies
-
- The microorganisms harvested from each culture were washed
once in
- phosphate buffered saline (PBS) and then resuspended
in PBS. Protein
- concentrations of each suspension were determined using
the Bio-Rad protein
- assay kit (Bio-Rad instruction manual). Antigenic analysis
with polyclonal
- and monoclonal antibodies was done using the Bio-Dot
microfiltration
- apparatus (Bio-Rad).
-
- One hundred microliter samples from each dilution which
contained
- decremental amounts (either 1:4 or 1:10 dilution in PBS)
of proteins were
- dot-blotted onto nitrocellulose paper under vacuum. The
blots were blocked
- with 5% non-fat milk and reacted with either M. fermentans
incognitus
- specific rabbit antiserum (1:1000 in PBS) (Lo, S-C et
al., Am. J. Trop. Med
- Hyg. 40, 215 (1989)), or M. fermentans incognitus specific
mule antiserum
- (1:4000 in PBS), provided by Dr. Richard A. Dol Guidice
of Frederick, Md.
- The titers of the rabbit M. fermentans incognitus antiserum
and the mule M.
- fermentans incognitus antiserum had previously been determined
to be 20,000
- and 80,000, respectively.
-
- The blots were then reacted with biotinylated goat antirabbit
IgG (Vector)
- and biotinylated goat antihorse IgG (Vector), respectively.
In the
- antigenic analysis using monoclonal antibodies, the concentration
of
- primary antibody was adjusted to 20 fold of each monoclonal
antibody titer.
- The titers of these monoclonal antibodies were previously
determined to be
- D81E7, 5.1.times.10.sup.4 ; C69H3, 2.6.times.10.sup.4
; F89H7,
- 2.0.times.10.sup.5 ; B109H8, 2.6.times.10.sup.4 ; F11C6,
6.4.times.10.sup.3
- ; and C24H10, 2.6.times.10.sup.4. The biotinylated horse
antimouse IgG or
- goat antimouse Igm (Vector) were used as the secondary
antibodies according
- to the specific isotype of each monoclonal antibody.
Each incubating step
- was conducted for 30 minutes at room temperature with
three Tris buffered
- saline-Tween 20 (0.2%) washes between steps. The color
reaction was
- developed in diaminobenzidine and H.sub.2 O.sub.2 substrate
after formation
- of avidin-biotin complex.
-
- Both biological characterization and DNA homology analysis
indicated that
- M. fermentans incognitus was distinct from all other
species of human
- mycoplasmas, but closely related to M. fermentans incognitus.
Therefore, a
- detailed comparison between these two species was performed
by studying
- their specific antigenicity.
-
- Polyclonal rabbit antiserum raised originally against
VLIA-Sb51 (Lo, S-C et
- al., Am. J. Trop. Med. Hyg. 40, 339 (1989)) was found
to react with M.
- fermentans in addition to M. fermentans incognitus, but
not with any other
- mycoplasmas examined (FIG. 2A). However, a larger amount
of M. fermentans
- protein (>0.63 mg) was required to elicit the positive
immunochemical
- reaction in this assay. The positivity of reaction quickly
disappeared when
- the M. fermentans proteins were further diluted. In comparison,
a 250-fold
- to 1000-fold lower concentration of M. fermentans incognitus
proteins still
- carried a sufficient amount of antigenic determinants
and exhibited
- positive reactions in the assay (FIG. 2A).
-
- In the parallel assay, antiserum raised specifically
against M. fermentans
- also reacted intensely with M. fermentans incognitus
(FIG. 2B). The M.
- fermentans-specific antiserum appeared to cross react
with A. laidlawii and
- M. orale when high concentrations (10 mg) of mycoplasma
proteins were
- dot-blotted. M. fermentans antiserum reacted with the
antigens of M.
- fermentans incognitus proteins. Both M. fermentans incognitus
and M.
- fermentans proteins could be diluted to 40 ng per well
and still elicit a
- positive reaction (FIG. 2B).
-
- FIG. 2 shows antigenic comparison of M. fermentans incognitus,
M.
- fermentans and other human mycoplasmas in immunoblots.
Upper blot (A) was
- immunostained with rabbit antiserum raised specifically
against M.
- fermentans incognitus. Lower blot (B) was immunostained
with rabbit
- antiserum raised specifically against M. fermentans.
The concentration of
- mycoplasma protein was dot-blotted decrementally (1:4
dilution) from lane 1
- (10 mg) to lane 12 (2.5 pg). Row A (M. arginini), row
B (A. laidlawii), row
- C (M. fermentans), row D M. hominis), row E (M. orale),
row F (M.
- hyorhinis), row G (M. pnuemonia), row H (M. fermentans
incognitus). In FIG.
- 2 (C) row A, B, C, D and F were immunostained with monoclonal
antibodies
- D81E7, C69H3, F89H7, B109H8, F11C6 and C42H10, respectively.
The
- concentration of mycoplasma protein was dot-blotted decrementally
(1:10
- dilution) from lane 1 (10 ug) to lane 8 (1 pg). Row a
(M. fermentans
- incognitus) and Row b (M. fermentans).
-
- In order to examine the possibility suggested by the
above results that M.
- fermentans incognitus carried additional unique antigens
which are not
- present in M. fermentans, a battery of monoclonal antibodies
raised
- specifically against M. fermentans incognitus were prepared.
All six M.
- fermentans incognitus monoclonal antibodies obtained,
many with different
- isotypes, were found to react only with M. fermentans
incognitus but not
- with M. fermentans (FIG. 2C). These monoclonal antibodies
also did not
- react with any of the other nine Mollicutes examined.
- Table 7 summarizes the results of the antigenic analysis
using both
- polyclonal and monoclonal antibodies. The results confirmed
that M.
- fermentans incognitus carries additional specific antigens
which could not
- be identified in M. fermentans.
-
-
- TABLE 7
- __________________________________________________________________________
- Characterization and Comparison of Antigenicity Between
Mycoplasma
- incognitus and Seven Other Mollicutes
- Species
- ANTIBODIES
- ISOTYPE MA AL === MHO MO MHY MP
===
- __________________________________________________________________________
- Rabbit antiserum
- Polyclonal
- - - - - - - -
+++
- Against MI
- Mule antiserum
- Polyclonal
- - .+-.
- +++ - .+-.
- - -
+++
- against MF
- D81E7 Monoclonal
- - - - - - - -
+++
- IgM/K
- C69H3 Monoclonal
- - - - - - - -
+++
- IgM/K
- F89H7 Monoclonal
- - - - - - - -
+++
- IgM/K
- B109H8 Monoclonal
- - - - - - - -
+++
- IgG3/K
- F11C6 Monoclonal
- - - - - - - -
+++
- IgG3/K
- C42H10 Monoclonal
- - - - - - - -
+++
- IgG1/K
- __________________________________________________________________________
- Labels +++, +, .+-. and - denote the relative positivity
of immunostainin
- results in FIG. 4. MA: M. argini, Al: A. laidlawii,
MF: M. fermentans,
- MHO: M. himinis, MO: M. orale, MHY: M. hyorhinis,
MP: M. pneumoniae, MI,:
- M. incognitus
-
-
- Direct immunofluorescense examination
-
- Monoclonal antibody was purified from ascites fluid by
high-salt
- precipitation and gel chromatography using Sephacryl-200
(Campbell, D. H.
- et al., Method in Immunology 2d Ed., W. A. Benjamin,
Inc., p. 198 (1970)).
- Labeling of the purified antibody with fluorescein isothiocyanate
(FITC)
- was done using the method described by Rinderknecht (Nature
193, 167
- (1962)). The broth culture suspensions were directly
smeared on the slides.
- The slides were air dried, fixed in 70% acetone, 30%
methanol and stored at
- 4.degree. C. The slides were directly immunostained with
FITC conjugated
- monoclonal antibody and examined under a fluorescent
microscope.
-
- In this study of direct immunofluorescense staining,
the FITC probe
- conjugated to the purified M. fermentans incognitus monoclonal
antibodies
- which again revealed positive staining only in M. fermentans
incognitus,
- but not in the other seven species of human mycoplasmas,
including M.
- fermentans (FIG. 4).
- FIG. 4 shows direct immunofluorescence staining of M.
fermentans incognitus
- (A) and M. fermentans (B) using FITC conjugated monoclonal
antibody D81E7
- (X900).
-
-
- EXAMPLE 23
-
- Identification of M. fermentans incognitus Infection
in Patients with Aids
-
- Monoclonal antibodies developed against antigens from
a pure culture of M.
- fermentans incognitus grown in modified SP-4 medium were
used to
- immunohistologically identify M. fermentans incognitus
infection in tissues
- of thymus, liver, spleen, lymph node or brain from 26
out of 32 patients
- with AIDS. M. fermentans incognitus infection was also
identified in 2
- placentas delivered by 2 patients with AIDS. The 32 patients
tested were
- homosexuals, intravenous drug abusers or pediatric patients
who had
- received transfusions.
-
- M. fermentans incognitus specific DNA was also identified
in the subject
- tissues using a .sup.35 S labeled psb-2.2 DNA probe and
in situ
- hybridization. Although M. fermentans incognitus was
found to be both
- cytopathic and cytocidal, the cellular immune response
and inflammatory
- reaction to M. incognitus infection was often atypical.
-
- Patient profiles
-
- All 34 AIDS patients were selected according to CDC criteria
(JAMA 258,
- 1143 (1987)). All patients were seropositive for HIV-related
antigens.
- Unless specified below, all the patients belonged to
the homosexual high
- risk group.
-
- Four thymic tissues, 10 livers from patients with unexplained
abnormal
- liver function tests, 8 spleens and 8 brains from patients
with clinicalCNS
- symptoms obtained at autopsy as well as 2 biopsied lymph
nodes were used.
- Histopathological studies using special tissue stains
did not reveal any
- bacterial, fungal or viral infectious agent in these
tissues. All tissues
- obtained at autopsy and biopsy were previously fixed
in 10% buffered
- formalin and embedded in paraffin blocks. Tissues of
non-AIDS control
- subjects were also obtained from 10% formalin fixed and
paraffin embedded
- blocks of autopsy tissues.
-
- Immunohistochemisty and in situ hybridization
-
- Deparaffinized and frozen section slides were incubated
with 10% bovine
- serumalbumin (Sigma Chemical Co.) in phosphate-buffered
saline (PBS, Gibco
- Co., pH 7.4 saline) for 30 minutes, rinsed briefly with
PBS, and covered
- with monoclonal antibodies (1:600 dilution).
-
- Slides were kept refrigerated overnight. After returning
to room
- temperature, the slides were rinsed with 1% albumin in
PBS. The slides were
- then covered with secondary antisera. Biotinylated horse
anti-mouse IgG
- (H&L) or biotinylated goat anti-mouse IgM (H&L)
(Vector Lab., Burlingame,
- Calif.) was added at 1:200 dilution in PBS as secondary
antisera and
- followed by the avidin biotinylated peroxidase complex
(ABC) reagent
- (Vector Lab, Burlingame, Calif.). Each incubation step
was conducted for 1
- hour with extensive washing between steps. The color
reaction was developed
- in DAB-H.sub.2 O.sub.2 substrate and counterstained with
hematoxylin.
-
- Development of M. fermentans incognitus-specific monoclonal
antibodies
- (C42H10, and D81E7) has been described previously in
Example 21. In
- parallel, non-specific mouse monoclonal antibodies (IgM,
MOPC 104E and
- IgG.sub.2b K, MOPC 141, Sigma) or monoclonal antibody
(ascites) raised
- specifically against herpes virus (IgG.sub.1, MCA 255,
clone R1,
- Bioproducts) were used as the primary antibodies and
served as negative
- controls in immunohistochemistry. Detailed procedures
of preparation of
- .sup.35 S radiolabeled psb-2.2 probe and in situ hybridization
on
- formalin-fixed and paraffin embedded tissues were also
described previously
- (Lo, S-C et al., Am. J. Trop. Med. Hyg. 41, 380 (1989)).
-
- Electron microscopy
-
- To retrieve formalin-fixed paraffin-embedded tissues
for ultrastructural
- examination, immunohistochemistry positive areas of tissue
sections on
- glass slides were circled. These exact area were then
matched and
- identified on each individual paraffin block. Tissues
of 1 to 2 mm in
- diameter were punched out from the blocks and deparaffinized
in xylene.
- Processing of these tissues for E. M. studies were previously
described in
- detail (Lo, S-C et al., Am. J. Trop. Med. Hyg. 41, 380
(1989)).
-
- RESULTS
-
- Thymus
-
- Many patients with AIDS suffer a profound deficiency
in cell mediated
- immunity. It is well known that development of competent
T-cell immunity is
- thymus dependent. Therefore, four thymic tissues available
from patients
- with AIDS were examined for possible M. fermentans incognitus
infection.
- Two of the thymic tissues were described grossly at autopsy
as involuted
- thymus, one from a two year old and the other from a
eight year old. Both
- of these pediatric patients contracted AIDS from blood
transfusions.
- The other two thymuses were derived from adult AIDS patients
and the
- autopsy reports contained no specific gross tissue description.
- Immunohisto-chemical studies, using M. fermentans incognitus-specific
- monoclonal antibodies, showed positive immunoreaction
in all four thymic
- tissues. Both mononuclear lymphohistiocytes and epitheloid
cells were
- stained positively (FIG. 26).
-
- FIG. 26 shows the immunhoistochemistry of thymic tissues
derived from
- patients with AIDS. FIG. 26A is a low-magnification photograph
of a thymus
- immunostained by M. fermentans incognitus-specific monoclonal
antibody
- (C42H10) (X71.5). FIG. 26B is a higher magnification
of the positively
- immunostained lymphohistiocytes in the junction between
cortex and medulla
- shown in 26A, left curve arrow (X715). FIG. 26C is a
higher magnification
- of the positively immunostained lymphohistiocytes in
the septal
- interstitial tissues in 26A, right curve arrow (X715).
FIG. 26D is a
- low-magnification photograph of a thymus from another
AIDS patient
- (X126.5). FIG. 26E is a higher magnification of the positively
- immunostained cells in 26D (X142).
-
- Electron microscopic examination of the areas of the
thymus with
- significant positive immunoreaction showed ultrastructurally
many particles
- resembling mycoplasma. The particles were located both
intracellularly in
- the cytoplasm of lymphohistiocytes (FIG. 27 A, B) and
apparently
- free-growing extracellularly (FIG. 27 C, D). FIG. 27
shows an electron
- micrograph of an AIDS thymus immunostained positively
for M. fermentans
- incognitus-specific antigens. FIG. 27A is an electron
micrograph of
- mononuclear lymphohistiocytes with many intracytoplasmic
electron dense
- mycoplasma-like particles (arrows) (N is the nucleus
and bar represents 100
- nm). FIG. 27B is a higher magnification of the electron
dense
- mycoplaslma-like particles in the cytoplasm of a mononuclear
cell shown in
- 27A (P is a polysomal structure and bar represents 100
nm). FIG. 27C is an
- electron micrograph of many mycoplasma-like particles
found both inside the
- membrane bound cytoplasmic vesicle (arrow heads) and
also extracellularly
- in the interstitial tissue (arrows) (N is the nucleus
with degenerating
- changes, Bar represents 100 nm). FIG. 27D is a higher
magnification of the
- extracellular mycoplasma-like particles. The outer limiting
membrane of
- some particles (arrows) can be identified (Bar represents
100 nm).
-
- Most of the nearly spherical particles measured 100-300
nm. No cell wall
- was associated with these particles. However, a prominent
halo with a clear
- space surrounding each of these intracellular particles
was commonly noted.
-
- Occasional cells exhibited cytopathological changes and
even appeared to be
- necrotic. However, most cells in these tissues were morphologically
- unremarkable. There was no tissues reactive process and/or
an inflammatory
- reaction identified.
-
- Liver
-
- Ten livers from patients with AIDS who had unexplained
abnormal liver
- function tests were examined. Work-ups for both hepatitis
B and A
- infections were negative in these patients.
-
- Four of these ten livers were positive by immunohistochemistry
using M.
- fermentans incognitus-specific monoclonal antibodies.
Histopathology of
- these four livers varied from no pathological changes
except mild
- periportal infiltrates of lymphohistiocytes (two) to
fulminant hepatocyte
- necrosis without any inflammatory reaction (one) and
patchy areas of
- hepatocyte necrosis associated with prominent acute and
subacute
- inflammation (one). The positively immunostained cells
in these livers were
- the infiltrating inflammatory cells and the hepatocytes
with or without any
- evidence of cytopathological changes (FIG. 28). Some
areas of sinusoidal
- space lined by Kupffer cells were also stained positively.
-
- FIG. 28 shows the immunohistochemistry of livers derived
from patients with
- AIDS, using monoclonal antibody C42H10. FIG. 28A is a
photomicrograph at a
- portal area in an AIDS liver with patchy areas of necrosis.
Prominent
- infiltrates of chronic inflammatory cells and proliferation
of bile ducts
- (arrows) are identified (X390). FIG. 28B is a higher
magnification of the
- positively immunostained cells in 28A (X780). FIG. 28C
is the same portal
- area shown in 28A in a subsequent tissue section immunostained
by a
- nonspecific monoclonal antibody with the same isotype
IgCl/k. Hemosiderin
- pigments (arrow heads) are noted (X390). FIG. 28D is
an immunohistochemical
- photomicrograph of another AIDS liver. No necrosis or
histopathological
- changes other than mild infiltrates of chronic inflammatory
cells in the
- portal area (P) can be found in the liver (X390).
-
- The areas of liver showing positive M. fermentans incognitus-
specific
- antigens were also retrieved from the original paraffin
blocks for ultra
- structural examination. Microorganisms with typical mycoplasma
morphology
- were identified in all four livers. These mycoplasma-like
microorganisms
- could be found intracellularly in the cytoplasms of mononuclear
- lymphohistiocytes, Kupffer cells and hepatocytes. Many
of these
- microorganisms also lined up extracellularly along the
walls of sinusoids
- (FIG. 29). For comparison, an electron micrograph of
M. fermentans
- incognitus in the liver of a silvered leaf monkey, experimentally
infected
- with this pathogen (Example 9) is shown in the insert
of FIG. 29E.
-
- FIG. 29 shows an electron micrograph of AIDS liver immunostained
positively
- for M. fermentans incognitus-specific antigens. FIG.
29A is an electron
- micrograph of a periportal area of an AIDS liver with
adjacent necrosis. N
- is the nucleus of a mononuclear lymphohistiocyte. R is
red blood cells in
- the small vessel and the bar represents 500 nm. FIG.
29B is a higher
- magnification of the mycoplasma-like microorganisms found
in the empty
- extracellular space and lining along the outer surface
of the
- lymphohistiocyte shown in 29A. Many intracellular particles
(arrow heads)
- can also be identified and are difficult to differentiate
with the
- extracellular particles (P is the polysomal structure
and the bar
- represents 1200 nm). FIG. 29C is a higher magnification
of the
- mycoplasma-like microorganisms lining the outer surface
of the
- lymphohistiocyte (Bar represents 100 nm). FIG. 29D is
an electron
- micrograph of another AIDS liver which showed no evidence
of
- histopathological changes except mild portal infiltrates
of chronic
- inflammatory cells (N is the nucleus and the bar represents
400 nm). FIG.
- 33E is a higher magnification of the mycoplasma-like
particles shown in
- 29D. The insert shows M. fermentans incognitus in 2%
glutaldehyde fixed
- liver of experimentally infected silvered leaf monkey
at the same
- magnification (Bar represents 100 nm).
-
- Lymph node and spleen
-
- Two lymph nodes surgically removed from AIDS patients
showed reactive
- changes with follicular hyperplasia and foci of sinus
histiocytosis. No
- areas of necrosis were identified. Positive immunochemical
reactions were
- seen primarily within the endothelial cells lining the
lymphatic sinus or
- the mononuclear lymphohistiocytes found in the sinus.
Both nuclei and
- cytoplasm were stained positively. The typical staining
patterns were
- similar to the results presented previously, using polyclonal
rabbit
- antiserum (Lo, S-C et al., Am. J. Trop. Med. Hyg. 40,
213 (1989)).
-
- Sections from four of six autopsy spleens without pathological
changes
- stained positively with M. fermentans incognitus-specific
- monoclonalantibody. Mononuclear histiocytes and reticular
cells in
- periarterial regions, mononuclear, reticular cells and
lymphocytes in areas
- of red pulps were the positive cells which often revealed
varying degrees
- of swelling or disruption. The strongly-stained nuclei
and cytoplasm
- resembled inclusion bodies in the immunochemical reaction.
Positively
- stained cells could also be identified in two additional
splenic tissues
- with areas of prominent necrosis. The positive immunochemical
reaction was
- concentrated at periphery of the necrosis (data not shown).
- Characteristic ultrastructures with morphological features
typical of
- mycoplasma were identified in all four spleens (including
two with
- necrosis) and two lymph nodes which were retrieved for
electron microscopy.
-
- Brain
-
- More than 60% of patients with AIDS are reported to have
abnormal central
- nervous system (CNS) symptoms (Navaia, B. A. et al.,
Ann. Neurol. 19, 517
- (1986)). Since most AIDS patients have serological evidence
of HIV
- infection, the CNS diseases in these patients with AIDS
have been called
- HIV encephalopathy.
-
- Eight brains from patients with AIDS who had prominent
clinical symptoms of
- CNS diseases without histopathological diagnosis of a
specific infection in
- the brains at necropsy were examined. Two of these 8
brains had lesions of
- fulminant necrosis and karyorrhexis associated with both
acute and subacute
- inflammations. Both of these brains were from intravenous
drug abusers with
- AIDS. One of the other brains had subacute encephalitis
with mononuclear
- cell infiltration but no necrosis. The remaining 5 brains
showed only
- atrophy, gliosis and occasional microglial nodules without
evidence of
- necrosis or inflammation.
-
- All 3 brains with histopathological evidence of acute
or subacute
- encephalitis stained positively for M. fermentans incognitus-specific
- antigens. FIG. 30 shows the positive immunostaining of
the acute and
- subacute inflammatory cells in the periphery of a necrotic
brain lesion.
-
- FIG. 30A is a photomicrograph of the periphery of a necrotic
cerebellar
- lesion immunostained positively by M. fermentans incognitus-specific
- monoclonal antibody (C42H10) (X390). FIG. 30B is a higher
magnification of
- the periphery of the lesion in 30A and shows both acute
and subacute
- inflammatory cells immunostained positively (X780). FIG.
30C is also a
- higher magnification of the positively stained cells
in 30A (X780). FIG.
- 30D is a photomicrograph of the same periphery area of
the necrotic lesion
- immunostained by a non-specific monoclonal antibody with
the same isotype
- IgG1/k. Cells with prominent cytopathological changes
and disruption
- (arrows) are evident (X780).
-
- Furthermore, three of the 5 brains showing no evidence
of inflammation or
- necrosis also revealed positive immunostaining. The positively
stained
- cells showed degenerating changes, and often became inclusion
body-like
- structures in the gray and white matter. The patterns
and characteristics
- of positive immunohistochemical staining identified in
these histologically
- unremarkable brains were comparable to those previously
reported, using
- rabbit polyclonal antiserum (Lo, S-C et al., Am. J. Trop.
Med. Hyg. 40, 213
- (1989)).
-
- Ultrastructural confirmation of M. fermentans incognitus
infection in these
- 6 brains which immunostained positively for M. fermentans
- incognitus-specific antigens was also performed. Many
electron-dense
- particles with features of mycoplasma organisms were
identified
- extracellularly positively for M. fermentans incognitus-specific
antigens
- was also performed. Many electron-dense particles with
features of
- mycoplasma organisms were identified extracellularly
or in the cytoplasm of
- mononuclear lymphohistiocytes located in the periphery
of necrosis.
- Clusters of particles with morphological features of
mycoplasma could also
- be identified in the encephalopathy AIDS brains showing
positive
- immunostaining but with no evidence of necrosis and inflammation
(FIG. 31).
- Some of the particles had prominent outer membranes.
For comparison, the
- electron micrograph of M. fermentans incognitus with
an apparent outer
- limiting membrane identified in cytoplasm of Sb51 cells
in culture is shown
- in the insert of FIG. 31D.
-
- FIG. 31A is an electron micrograph of mycoplasma-like
particles (arrows)
- clustered together in the hippocampus. F is a bundle
of neuroglialfilament
- and N is the nucleus of a mononuclear cell (Bar represents
100 nm). FIG.
- 31B is a higher magnification of the mycoplasma-like
particles shown in
- 31A.
-
- The outer limiting membrane (small arrows) of some particles
is prominent.
- (Bar represents 100 nm). FIG. 31C is a higher magnification
of the same
- particles. FIG. 31D is a high magnification electron
micrograph of
- mycoplasma-like particles found in the brain stem from
another AIDS patient
- (large photo to right). The typical particles with well-preserved
outer
- membrane (small arrows) are shown in an endothelial cell.
Cytoplasmic
- membrane (large arrows) of the endothelial cells and
basement membrane
- (arrow heads) of the vessel can be identified. L is the
lumen of the
- vessel. The insert shows an electron micrograph of VLIA
(M. fermentans
- incognitus) originally identified in the cytoplasm of
sb51 cells, at the
- same magnification. The unit membrane of M. fermentans
incognitus (small
- arrows) is prominent in the well fixed (2% glutaldehyde)
and well preserved
- culture specimen. Cytoplasmic membrane (large arrows)
of the sb51 cell is
- also identified (Bar represents 200 nm).
-
- Placentas
-
- Two placentas delivered at full term by two women with
AIDS were available
- for study. The babies were reported to be normal at birth.
However, no
- follow-up was available.
- Histopathological examination showed occasional infiltrate
of acute
- inflammatory cells in the chorionic plates in one of
the placentas. The
- second placenta was histologically unremarkable. The
special
- histopathological stains did not reveal any pathogens
in either of the two
- placentas. Immunohistochemical study of both placentas,
using M. fermentans
- incognitus-specific monoclonal antibodies C42H10 and
D81E7, exhibited
- positive immunoreaction in areas of Hofbauer cells and
stomal connective
- tissues in the chorionic villi (FIG. 32). Some decidual
cells in the
- stratum basalis were also stained positively.
-
- FIG. 32 shows the immunohistochemistry of a placenta
delivered by a patient
- with AIDS. FIG. 32A is a photomicrograph of placenta
tissue positively
- immunostained by a M. fermentans incognitus-specific
monoclonal antibody
- (C42H10). The insert shows the same placental area in
a subsequent tissue
- section immunostained by a non-specific monoclonal antibody
with the same
- isotype IgG1/k (X 195). FIG. 32B is a higher magnification
of the
- positively immunostained cells shown in 32A. The cytoplasm
(arrow heads) or
- the surface of vacuolated cells (arrows) more often reveals
positive
- reaction. Cells showing cytopathological changes with
both nuclei and
- cytoplasms are positively stained (curve arrows) may
resemble atypical
- inclusion bodies (X780).
-
- Electron microscopic examination of the Hofbauer cells
and connective
- tissues in the positively stained chorionic villi revealed
numerous
- particles characteristic of mycoplasma (FIG. 33). Some
particles identified
- in the Hofbauer cells were probably in membrane bound
vesicles.Many
- microorganisms, with a wide variation of size, shape
and electron density,
- appeared to focally colonize in the stomal connective
tissue (FIG. 33). A
- prominent halo with a clear space surrounding each of
these particles was
- often noted. No accompanying acute inflammatory cells
or other reactive
- process was identified. Some apparently better preserved
particles
- exhibited recognizable outer limiting membranes. However,
many of the
- mycoplasma-like particles did not have definite outer
unit membranes; they
- showed only an electron dense internal matrix with a
fine granular
- configuration.
-
- FIG. 33 shows electron microscopy of an AIDS patient's
placenta
- immunostained a positively for M. fermentans incognitus
specific
- antigens.FIG. 33A is an electron micrograph of a Hofbauer
cell containing
- may mycoplasma-like particles in the cytoplasm. Some
particles are
- apparently in the membrane bound cytoplasmic vesicles
(arrows). N is the
- nucleus and I is a cytoplasmic inclusion body (Bar represents
800 nm). FIG.
- 33B is a higher magnification of the mycoplasma-like
particles. Both
- spherical electron dense particles (arrow heads) and
flask shape particles
- (arrows) typical for mycoplasma organisms are found to
colonize in the
- stomal connective tissue (Bar represents 1000 nm). FIG.
33D is a higher
- magnification of the mycoplasma-like particles shown
in 33C. Typical
- electron dense internal matrix with fine granular configuration
of these
- particles is shown. Occasional particles contain recognizable
outer
- membrane (arrows) (bar represents 100 nm). FIG. 33E shows
many of the
- particles are also those of less electron dense but with
granular appearing
- internal matrix. These particles often have more prominent
outer limiting
- membrane (arrows) (Bar represents 100 nm).
-
- Detection of M. fermentans incognitus specific genetic
material
-
- M. fermentans incognitus DNA was identified in the tissues
of thymus, liver
- and spleen from patients with AIDS as well as in the
placentas delivered by
- two women with AIDS using the .sup.35 S labeled psb-2.2
probe. FIG. 34
- shows positive labeling with grains heavily concentrated
in cells of livers
- and spleen. Cytological and/or histological identification
of the specific
- "types" of cells containing M. fermentans incognitus
DNA, revealed that
- they were the Kupffer cells and hepatocytes in the liver
showing minimal
- histopathological changes (FIG. 34A), the infiltrating
lymphoid cells and
- histiocytes in portal tracts of another liver (FIG. 34C),
and the
- lymphocytes in periarteriolar lymphoid sheaths (white
pulp) of spleen (FIG.
- 34D).
-
- In parallel, .sup.35 S-labeled M13 mp 19 vector DNA which
did not contain
- M. fermentans incognitus DNA, did not elicit any positive
signals in the
- consecutive sections from these tissues (FIG. 34B). Five
tissues of spleen
- and liver from three patients who died of non-AIDS conditions
were used as
- negative controls and also did not reveal any evidence
of positive signals.
-
- FIG. 34 shows in situ hybridization for M. fermentans
incognitus nucleic
- acid in liver and spleen from patients with AIDS. FIG.
34A shows cells with
- strong labeling (arrows) are seen in an AIDS liver with
no
- histopathological abnormally after hybridization with
.sup.35 S labeled
- psb-2.2 DNA. Higher magnification (insert) reveals dense
clusters of grains
- over individual hepatocytes or Kupffer cells (X240, X770).
FIG. 34B is the
- same area of 34A in the consecutive tissue section, hybridized
with .sup.35
- S-labelled cloning vector DNA not containing M. fermentans
incognitus DNA
- (X270). FIG. 34C shows lymphocytes and histiocytes with
positive labeling
- seen in the portal tract infiltrated with mononuclear
inflammatory cells in
- the liver of another AIDS patient (X770). FIG. 34D shows
lymphocytes with
- strong labeling seen in the periarteriolar lymphoid sheath
of the spleen.
- The central arteriole (Ar) is identified. The insert
shows higher
- magnification of heavily concentrated grains over the
lymphoid cells in
- this white pulp
- (X350, X770).
-
- Kidney
-
- Renal tissues from 203 patients who died of AIDS as defined
by the Centers
- for Disease Control criteria were selected for study.
The patients lived in
- various geographic locations including the continental
United States (US),
- Puerto Rico (PR), Haiti, and Africa. The different racial
backgrounds
- included in this study were white, black, Hispanic, and
Oriental. Risk
- activities for AIDS were varied and included intravenous
drug abuse (IVDA),
- homosexual contact, heterosexual contact, and history
of blood transfusion.
- The patients had a wide range of opportunistic infectious
agent including
- Pneumocystis carinii, Toxoplasma gondii, Candida albicans,
Cryptococcus
- neoformans, Histoplasma capsulatum, Mycobacterium avium-intracellulare,
M
- tuberculosis, cytomegalovirus, herpes simplex virus,
and others.
-
- Of the 203 total patients comprising this study, 20 patients
had renal
- histopathologic changes characteristic of AIDS-associated
nephropathy
- (AAN). Group B consisted of 15 patients selected from
the remaining 183 who
- had no significant clinical or pathologic evidence of
renal disease. These
- patients were matched as closely as possible with Group
A patients in terms
- of the distribution of age, gender, race, and risk activities
which
- Sections of kidney from the autopsies of 203 patients
with AIDS, as well as
- renal tissues from the five (Group C) controls, were
examined by
- conventional light microscopy.
-
- Special stains, including periodic acid-Schiff, Grocott's
methenamine
- silver, Ziehl-Neelsen, mucicarmine, Masson's trichrome,
and Brown and
- Hopps, were obtained to evaluate glomerular and tubular
morphology as well
- as to document the presence of various opportunistic
infections. For the 20
- cases of AAN, glomerular, tubular, and interstitial changes
were
- semiquantitatively graded and recorded.
-
- Renal tissues from 15 of the 20 patients from Group A
and all of the
- tissues from Groups B and C were evaluated using monoclonal
antibodies
- (MABs) against M. fermentans incognitus as described
above.
-
- Formalin-fixed, paraffin-embedded sections of kidney
were immunochemically
- stained with MABs against the incognitus strain, as previously
described.
- Specific areas of positive staining were circled (approximately
1 mm in
- diameter) and removed from the matched paraffin tissue
blocks. Tissues were
- then deparaffinized and processed as described above.
After embedding all
- tissues in epoxy resin, semi-thin sections were cut and
stained with
- alkaline toluidine blue for histologic analysis. The
thin sections of the
- selected blocks were stained with lead citrate and uranyl
acetate and
- examined by electron microscopy.
-
- Light Microscopy
-
- For all 20 cases of AAN, the earliest recognizable glomerular
change
- consisted of relative and actual dilatation of Bowman's
space, with
- concomittant capillary tuft wrinkling, compression, or
complete collapse
- (FIG. 1a). Bowman's spaces often contained finely granular
proteinaceous
- material which was also present in the lumens of adjacent
proximal
- convoluted tubules. The subsequent glomerular change
was "early" segmental
- or global glomerulosclerosis, as evidenced by hypertrophy
and vacuolization
- of visceral epithelial cells and capillary endothelial
cells, increased
- amounts of mesangial matrix material in either a segmental
or global
- distribution, and small protein droplets within epithelial
cells and
- Bowman's space. The most advanced glomerular change was
fully evolved
- ("late") segmental and global sclerosis. In
the latter case, glomerular
- tufts were transformed to round "sclerotic balls,"
sometimes surrounded by
- a rim of hypertrophic epithelial cells. In this advanced
stage,
- homogeneous, dense cast material often filled the dilated
Bowman's spaces
- and contiguous tubular lumens. The peripheral edges of
these casts had
- scalloped borders, created by side by side "holes"
in the cast material
- adjacent to tubular epithelial cells.
-
- Tubular changes usually paralleled glomerular changes.
In early stages,
- tubular epithelial cells with cytoplasmic swelling contained
many protein
- droplets. Subsequently, tubular lumens became dilated
and contained protein
- droplets or granular proteinaceous material, as well
as degenerated
- sloughed epithelial cells. In later stages, tubules showed
microcystic
- dilatation and were filled by dense cast material. Epithelial
cells within
- such tubules were flattened from compression by the large
proteinaceous
- casts. In all cases, variable degrees of interstitial
edema and mononuclear
- cell inflammation were present. Special tissue stains
did not reveal any
- evidence of infection with bacteria, fungi, or mycobacteria
in these
- kidneys.
-
- Sections of renal tissue from the 15 group B patients
showed minimal
- structural abnormalities including focal mild mononuclear
cell infiltration
- of the interstitium, rare mononuclear cells within glomerular
capillary
- lumens, and occasional hyaline casts. Renal tissue from
three of the five
- group C patients also demonstrated normal histology,
renal tissue from the
- remaining two showed changes consistent with reflux nephropathy
and
- moderate to marked nephrosclerosis, respectively.
-
- Immunohistochemistry
-
- For all of the 15 group A patients, there was positive
staining by M
- incognitus-specific MABs in several locations including
glomerular
- endothelial and epithelial cells, capillary basement
membrane, tubular
- epithelial cells, tubular casts, and mononuclear interstitial
cells.
- Although all cases had positive staining for antigens
of this microorganism
- in the above locations, six cases showed more prominent
positivity in
- glomerular epithelial and endothelial cells, while nine
cases had greater
- positivity in tubular epithelium and casts. Particularly
intense staining
- could often be seen in partially degenerated cells within
the casts, or
- within the amorphous cast material itself.
-
- Kidney tissues from group B patients showed positive
staining for
- incognitus strain-specific antigens only within occasional
mononuclear
- interstitial cells. These mononuclear cells were either
histiocytes or
- lymphocytes. None of the cases in this group demonstrated
positivity within
- the glomerulus or tubules.
-
- The renal tissues of group C patients showed no staining
for incognitus
- strain-specific mycoplasmal antigens in any locations.
-
- Electronmicroscopy
-
- Electron microscopic examination of tissues from the
particular areas
- highly positive for incognitus strain-specific antigens
revealed structures
- strongly resembling mycoplasmal organisms in various
locations in all 15
- group A cases.
-
- In seven patients, mycoplasma-like structures (MLS) were
identified in
- glomerular endothelial cytoplasm and in the adjacent
capitallary basement
- membrane. such endothelial cells often displayed enlargement
and
- vacuolization, with MLS sometimes localized in clusters
within the
- vacuoles.
-
- Although 12 patients showed MLS within the glomerular
basement membrane,
- seven patients, with mor eintense immunoperoxidase staining
for the
- mycosplasmal antigens within this location demonstrated
greater involvement
- of the memberane on electron microscopy. Mycoplasma-like
structures could
- be seen in subendothelial, intramembranous, and subepithelial
locations
- with accompanying membranopathic changes. These changes
consisted of (1)
- small holes in the basement membrane surrounding intramembranous
MLS, (2)
- splits and large irregular defects in the membrane associated
with
- scattered MLS, (3) thickening of the membrane, associated
with
- intramembranous MLS, and (4) complete breaks in the basement
membrane in
- areas of heavy MLS infiltration.
-
- Mycoplasma-like structures were also present within visceral
epithelial
- cells which often displayed cytoplasmic degeneration,
vacuolization, and
- partial detachment from the underlying basement membrane.
In many instances
- these cells were completely detached from the basement
membrane, embedded
- in proteinaceous cast material within Bowman's space.
-
- Numerous MLS were likewise found within the contiguous
large proteinaceous
- casts in microcystically dilated tubules. Morphologically,
these particles
- varied from spherical electron-dense forms to large ovoid,
flask-shaped or
- undulating forms. My coplasma-like structures were present
in great numbers
- in detached, degenerated tubular epithelial cells, which
were often
- incorporated into the casts.
-
- Electron microscopic study of renal tissues of 10 of
the 15 group B cases
- showed occasional mononuclear interstitial cells containing
MLS. Group C
- cases displayed no MLS ultrastructurally. Glomerular
endothelial
- tubuloreticular inclusions were present in the 15 group
A and 10 group B
- cases, and were absent in the five group C cases.
-
- In this study, we hae identified mycoplasmal infection
of the parenchymal
- cells in kidneys of AIDS patients with typical histologic
changes of AAN.
- There is good correlation between the immunohistochemical
presence of the
- incognitus strain mycoplasmal antigens in visceral epithelial
and tubular
- epithelial cells demonstrating the cytopathic changes
typical of AAN, and
- the ultrastructural presence of MLS within the same critical
cells. The
- same correlation also holds true for other microscopic
locations, such as
- glomerular endothelial cells and renal tubular casts.
The ultrastructural
- finding of significant numbers of MLS within the glomerular
capillary
- basement membrane with evidence of membranopathic effect
can be of
- particular importance when considering the pathogenesis
of this
- nephropathy.
-
- In summary, this study documents a spectrum of renal
histopathologic
- changes which helps further delineate the morphogenesis
of AAN. The study
- has also demonstrated the mycoplasmal infection of glomerular
endothelium,
- epithelium, and basement membrane, as well as tubular
epithelium, in the
- kidneys of AIDS patients with AAN. Infection of these
functional
- parenchymal cells by M. fermentans (incognitus strain)
may have contributed
- to the development of glomerulosclerosis, proteinuria,
intratubular casts,
- and renal failure in these patients with AIDS.
-
-
- EXAMPLE 24
-
- Enhancement of HIV-1 Cytocidal Effects in CD4.sup.+ by
M. fermentans
- incognitus
- The effects of the M. fermentans incognitus on HIV-1
infection of a
- CD4.sup.+ human T lymphocyte cell line, designated previously
as A3.01
- (Folks, T. et al., Science 231, 600 (1986)).
-
- Normally, HIV-1 infection of human T lymphocytes in vitro
produces
- pronounced cytopathic effects (CPE) with the release
of newly replicated
- virus (Lifson et al., Science 232, 1123 (1960)). The
formation of large
- multinucleated cells, termed syncytia, and high levels
of reverse
- transcriptase (RT) activity is a characteristic feature
of HIV-1 infection
- in vitro (Lifson et al., Nature 323, 725 (1986)). A3.01
cells
- (5.times.10.sup.7) were infected with () HIV-1 (1.times.10.sup.5
infectious
- units) and incognitus strain (1.times.10.sup.3 infectious
units), () HIV-1,
- or (0) incognitus strain. Cells in each culture were
incubated at
- 37.degree. C. for 2 hours and then washed once with RPMI
1640 medium. The
- infectious titer of HIV-1 was previously determined by
exposing A3.01 cells
- to tenfold serial dilutions of HIV-1 culture stock for
2 hours at
- 37.degree. C. The highest dilution in which the presence
of RT activity
- could be detected after 14 days in culture represented
one infectious unit.
- We grew the incognitus strain in modified SP-4 media
and filter-cloned it
- three times from a single colony on agar plates (3).
The organisms were
- washed once and resuspended in RPMI 1640. The titer of
incognitus strain
- after infection of NIH 3T3 cells was determined by antigen
dot blot assay.
- The cell cultures were maintained with RPMI 1640 supplemented
with 10% FBS.
- Large numbers of syncytia formed when HIV-1 alone infected
A3.01 cells, but
- syncytium formation disappeared in A3.01 cells simultaneously
infected with
- HIV-1 and incognitus strain (FIG. 35) despite clear evidence
of a cytocidal
- effect. Results are the average of the number of syncytia
per field (X200)
- of ten fields examined per culture. The error bars indicate
standard
- deviation of the mean.
-
- The cytocidal effect and inhibition of RT activity in
HIV-1 infected A3.01
- cell cultures by M. fermentans incognitus was analyzed.
A3.01 cells were
- cultured after () infection by HIV-1, () infection by
HIV-1 and incognitus
- strain, (.DELTA.) infection by incognitus strain, or
(*) no treatment. Each
- point on each graph is the average of the results of
three indipendent
- cultures. (A) Cell viability was determined with the
Trypan blue exclusion
- test with a total of 200 cells counted for each time
point. (B) Samples of
- culture supernatants were tested daily with the standard
RT enzyme assay
- using the incorporation of tritiated triphosphate nucleotides
(Baltimore et
- al., Proc. Natl. Acad. Sci. USA 68, 1507 (1971). Conditions
of HIV-1 and
- mycoplasma infectious were the same as described above.
The culture
- infected by mycoplasma alone [indicated by .DELTA. in
(A)] also had no
- detectable RT activity. The M. fermentans incognitus
significanly enhanced
- the cytocidal effects of HIV-1 infection in A3.01 cells
(FIG. 36A).
- Furthermore, populations of cells that had been infected
by HIV-1 alone
- gradually recovered from the initial cytocidal effect
and remained
- persistently infected. In contrast, A3.01 cells infected
by both HIV-1 and
- incognitus strain died. In this study, incognitus strain
infection alone
- did not produce detectable cytotoxicity. As expected,
culture supernatants
- from A3.01 cells infected with HIV-1 had clear RT activity.
However,
- samples from the cointected cell culture shoed little
or no RT activity
- (FIG. 36B).
-
- Despite the absence of RT activity, virus-specific protein
synthesis and
- assembly was occurring. This activity was shown by examining
culture
- supernatants. Culture supernantant (100 ul) was tested
for the presence of
- viral antigen (HIV-1 antigen assay kit, Integrated Diagnostics,
- Gaithersburg, Md.). The assay kit uses an enzyme-linked
immunosorbent assay
- (ELISA) technique, and the procedures performed in this
study were in
- strict accordance to the instructions supplied with the
kit. The negative
- control (phosphate-buffered saline) had an absorbance
(A.sub.410) reading
- of less than 0.1 at 410 nm. Each point on the graph (FIG.
37A) is the
- average of the results of three independent cultures.
()
-
- A3.01+HIV-1, (.DELTA.) A3.01+HIV-1+incognitus strain,
(*) A3.01 (FIG. 37B
- shows an electron micrograph of a cell culture infected
simultaneously with
- both HIV-1 and incognitus strain. Numerous viral particles
are seen in this
- culture with lytic cells. Occasional electron-dense forms
of incognitus
- strain (arrows) can also be seen. Bar+400 nm. The coinfected
cell culture
- produced HIV-1-specific p24-p25 as rapidly as the culture
infected by HIV-1
- alone (FIG. 37A). Electron microscopy of coinfected cells
showed typical
- HIV virions (FIG. 37B). The assembled virions were infectious.
Supernatant
- from the coinfected culture, which showed no detectable
RT activity, was
- tenfold serially diluted and incubated with fresh A3.01
cells. We found
- comparable infectious units of HIV-1 (10.sup.5 per milliliter)
to be
- produced in the supernatants after infection of cell
cultures either by
- HIV-1 alone or by both HIV-1 and incognitus strain (See,
Lo et al., Science
- 251, 1074 (1991).
-
- To test if substances in cultures infected by incognitus
strain directly
- affected the RT enzyme assay, culture supernatant from
A3.01 cells
- coinfected with HIV-1 and incognitus strain was mixed
with the culture
- supernatant containing HIV with known RT activity. Over
90% of the Rt
- activity was inhibited when less than a third of the
active supernatant was
- replaced by culture supernatants containing both HIV-1
and incognitus
- strain. Enzyme inhibition occurred immediately, and prior
incubation of the
- mixture of culture supernatants was not required. We
observed a comparable
- degree of inhibition when we used culture supernatant
from A3.01 cells
- infected with only incognitus strain in the inhibition
assay. Thus, the
- results can be best explained by the presence of some
mycoplasma product or
- products in the assay lysate which directly interfered
with the RT assay.
- Some mycoplasmas have recently been found to produce
highly active
- nucleases (Marcus et al., J. Cell Physiol 143, 416 (1990),
which could
- potentially be involved.
-
- The masking effect of HIV RT activity may not be unique
to incognitus
- strain. Suppression of HIV RT has recently been reported
in M.
- hyorhinis-contaminated lymphocyte cultures (Vasndevachari
et al., AIDS Res.
- Hum. Retroviruses 6, 411 (1990). But in contrast to the
results in this
- report, the HIV-1-infected cultures contaminated by the
swine mycoplasma
- still formed prominent syncytial cells. Our study indicates
that syncytium
- formation and the actual cytocidal effect can be separate
events. Our
- findings support the earlier reports (Sochoski, et al.,
Nature 322, 470
- (1986); Somasundaran, et al., J. Virol 61, 3114 (1987)
that state that the
- formation of syncytial cells is not a necessary prerequisite
for
- proliferation of HIV-1.
-
- It has recently been shown that nontoxic doses of the
antibiotic
- tetracycline may significantly reduce the cytocidal effects
of HIV-1
- (Lemaitre, et al., Res. Virol. 141, 5 (1990). The tetracycline-treated
- cultures continued to produce a high titer of HIV-1.
The authors suggested
- that a prokaryotic agent, most likely a mycoplasma, was
involved with the
- cytocidal effect observed in the HIV-infected cultures.
Indeed, additional
- study and characterization from their laboratory has
confirmed that the
- hidden agent in the cultures is a mycoplasma (Wright,
Science 248, 682
- (1990).
-
- Researchers from Japan have reported that just the antigens
of killed
- mycoplasma (Acholeplasma laidlawii) could stimulate HIV-1
production (p24
- antigen and infectious particles) in HIV-1-infected cells
(Chorodhurg et
- al., Lancet 336, 247 (1990). In our study, approximately
equivalent amounts
- of HIV antigen or infectious particles were produced
in HIV-infected or HIV
- and incognitus strain-infected cultures despite significant
differences in
- the numbers of viable cells. Thus, more HIV-1 may actually
have been
- produced per individual cell in the coinfected culture;
this finding is
- similar to the findings of the Japanese researchers.
-
- AIDS patients can be infected with a number of pathogenic
mircrobes and
- frequently are systemically infected with the incognitus
strain (Lo et al.,
- Am. J. Trop. Med. Hyg. 40, 213 (1989); Lo et al., Ibid
41, 601 (1989).
- Thus, the observation that coinfection by incognitus
strain profoundly
- enhances cytocidal effects of HIV-1 infection in vitro.
-
- While the invention has been described in connection
with specific
- embodiments thereof, it will be understood that it is
capable of further
- modifications. The description of the invention is intended
to cover any
- variations, uses or adaptations of the invention following,
in general,the
- principles of the invention, and includig such departures
from the present
- disclosure as come within known and customary practice
within the art to
- which the invention pertains.
-
-
- __________________________________________________________________________
- SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 17
- (2) INFORMATION FOR SEQ ID NO:1:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 22 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RS 48 Probe
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- GTTAGTTTTGGCATAAATCCCC22
- (2) INFORMATION FOR SEQ ID NO:2:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 2210 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasmaa fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: psb 2.2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- GAATTCTTTAATTGAGTTGCTCATTCTTGTTTCTTGAGTTTCAGTTAGTTTTGGCATAAA60
- TCCCCCCTTGTTTTTTATATTTAAATTATACTTTAAAGATTGTTAAAAAAACAATCATAT120
- GATTGTTTTAGAGTGAACCCCAAATTCCGGACTTTTTGGA AAGGGGTTCATTTTTATGCA180
- ATTTAAATTTAAAAAAGTAAAAAGAAACAAATGAAATAGAGATATAAAAGGTTATTTAAA240
- ATTAAAACTTGATCAAAAGATAAAAATTATCGAGTTATATTTTCAAGAATTTAGTATTTT300
- AGAAATATCTAAAATAAT GGAAAACTCTTATTCAGCATGCTATTCAGTAATAGAAAAATA360
- CAAAAAGTTTGGTTATAATTCTTTTGCTATGGAAAAGAAAAAAGGAAGAAAATCTAAAAT420
- AAATTTAGATGCTCAAAAGGCAACAAATTTTAAAATCAATATTGAAAATAAAATAGAAAA
480
- TAAAGATTTATTAATTAAACAATTAAAGGAAGAAAATAAAATACTCAAATTGGAGAATGC540
- GATAGCAAAAAAAGTGAGCGCCTTGGTTCAATTGAAAGACTCACTAACAAAGAAAAATTC600
- CAAATAACAATTGAACTAAGGCAAGAATTTAAAAAGCTAT TTTTTATTAAATTAATATTA660
- GAAAAAATTAAATTGAAAAAGTCAACTTTTTATGAGATATTAAAATCACAAAATAAACCT720
- GATAAAGATGAAAATTTAAAAAAGGTTATTTTTGACTTATTTAACTATAATAAAGGACTA780
- TACGGTTATAGACGTATT ACTTTTGCTTTAAGAAATAAAGGAATAATAATCAATCATAAA840
- AAAGTTCAAAAATTAATCGAAAGCAATGAATATTTTCGGCAAAACGCTAAGAAGAAAAAA900
- TAAATATTCTTCATTCAAAGGTGATGCTCACAAAACATTCCAAACTTGCTTTTAGATAAA
960
- GAAATATCACAGAAGATTTCTTCAGATACAAAAGAAATTTTTCAAATAATAAATATTTGA1020
- AAATACTAGGAACAGATGTTACTGAATTTAAATTAAAAAATGATGAAAAAGCATATTTTT1080
- CTCCTGTAGTTGATTTTGAAAACAGAGAGATTTTAGGTTA TTCGATTTCTAAATCGCCTA1140
- ATTTAAGAATGGTTGGTAAAATGTTAGAAAACGTAGAAGAGAATGGCCACAGCTTAAAAA1200
- ATGTATTATTACATTCTGATCAAGGATGACAATACACTCATCAAGATTATATTGATTATT1260
- TGAAAGAAAAACAAACAA CTCAAAGCATGTCAAGAAAGGGAAATTGTTTAGACAATAGTC1320
- CTACTGAATGTTTATTTAGTGTTATAAAAAGAGAATTTTGATTTGGAGAAGAAAAGAAAT1380
- TTAATAGTTTTAAAGAATTTAAAACTGCTTTAGGAGATATATTTCATATTATAATAATGA1
440
- CAGAATTGTTAATAAATTAAAAGACTTAGTCCTGTCCAATACAGGAATAAGTCCAAACAT1500
- AATTAAAAAGTCCAATTTTTGGGGTTCATACCATTTTGTGGAATTTTTCTTTTTTGCCAA1560
- TTTTTACCAAAGCACTATAAAACAGGCTTTTTAGAATTTT TCAAGCATTTCCATTTGTTT1620
- TTTAGGATATTTTTTAAATCGCAAATTTAACAAATTTTCTTATAGATGCTTCTATTTCTT1680
- GTTCTGATTTTTTAAGACCTATTTTTTTGATTAAACCATATTCAATGAAAAATAAAATTA1740
- ATAAATAAAGAGAAAGAA TTGTGAGTATTGAAAAGACACAAATTAAAACTCAAAGTAAAG1800
- TTGTATATGTGATTGATGGTGCCGCTTTATTTTGTCAAGCATAAGCGATTACAGTTATGA1860
- TCAATAGAATTATCATAAAAATAAATAGGAGTCCAAAAGCTTTAATATTCATTTGATTTC1
920
- TAAGATTTAAATGATCTAAATTGCTTTTGTACACTTTTTTATAAGCTTCTACTTTTTCTT1980
- CAAAAGAATATTTTTTCTTTTGCGTTTTTTATTTCTTGATCCATAACTTTCTCCTAATCA2040
- AAAGTAACATTCTTTAAGTTTTTGATTCAATTCAATATAT ATTTATATGTTCGGTCAAAA2100
- TCTATTTTTTTATCAACTTTAAAGTTTTTATTATCAGCAATTTGAGCTTCTATGTTATAA2160
- GCTTCAGTTTCGCTCAAATCATCCTTTGATTCAATATCAATATTGAATTC2210
- (2) INFORMATION FOR SEQ ID NO:3:
- (i ) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1405 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: IS element
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- TAGAGTGAACCCCAAATTCCGGACTTTTTGGAAAGGGGTTCATTTTTATGCAATTTAAAT60
- TTAAAAAAGTAAAAAGAAACAAATGAAATAGAGATATAAAAGGTTATTTAAAATTAAAAC120
- TTGATCAAAAGATAAAAATTATCGAGTTATATTT TCAAGAATTTAGTATTTTAGAAATAT180
- CTAAAATAATGGAAAACTCTTATTCAGCATGCTATTCAGTAATAGAAAAATACAAAAAGT240
- TTGGTTATAATTCTTTTGCTATGGAAAAGAAAAAAGGAAGAAAATCTAAAATAAATTTAG300
- ATGCTCAAAAG GCAACAAATTTTAAAATCAATATTGAAAATAAAATAGAAAATAAAGATT360
- TATTAATTAAACAATTAAAGGAAGAAAATAAAATACTCAAATTGGAGAATGCGATAGCAA420
- AAAAAGTGAGCGCCTTGGTTCAATTGAAAGACTCACTAACAAAGAAAAATTCCAAAT
AAC480
- AATTGAACTAAGGCAAGAATTTAAAAAGCTATTTTTTATTAAATTAATATTAGAAAAAAT540
- TAAATTGAAAAAGTCAACTTTTTATGAGATATTAAAATCACAAAATAAACCTGATAAAGA600
- TGAAAATTTAAAAAAGGTTATTTTTGACTTATTT AACTATAATAAAGGACTATACGGTTA660
- TAGACGTATTACTTTTGCTTTAAGAAATAAAGGAATAATAATCAATCATAAAAAAGTTCA720
- AAAATTAATCGAAAGCAATGAATATTTTCGGCAAAACGCTAAGAAGAAAAAATAAATATT780
- CTTCATTCAAA GGTGATGCTCACAAAACATTCCAAACTTGCTTTTAGATAAAGAAATATC840
- ACAGAAGATTTCTTCAGATACAAAAGAAATTTTTCAAATAATAAATATTTGAAAATACTA900
- GGAACAGATGTTACTGAATTTAAATTAAAAAATGATGAAAAAGCATATTTTTCTCCT
GTA960
- GTTGATTTTGAAAACAGAGAGATTTTAGGTTATTCGATTTCTAAATCGCCTAATTTAAGA1020
- ATGGTTGGTAAAATGTTAGAAAACGTAGAAGAGAATGGCCACAGCTTAAAAAATGTATTA1080
- TTACATTCTGATCAAGGATGACAATACACTCATC AAGATTATATTGATTATTTGAAAGAA1140
- AAACAAACAACTCAAAGCATGTCAAGAAAGGGAAATTGTTTAGACAATAGTCCTACTGAA1200
- TGTTTATTTAGTGTTATAAAAAGAGAATTTTGATTTGGAGAAGAAAAGAAATTTAATAGT1260
- TTTAAAGAATT TAAAACTGCTTTAGGAGATATATTTCATATTATAATAATGACAGAATTG1320
- TTAATAAATTAAAAGACTTAGTCCTGTCCAATACAGGAATAAGTCCAAACATAATTAAAA1380
- AGTCCAATTTTTGGGGTTCATACCA 1405
- (2) INFORMATION FOR SEQ ID NO:4:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 29 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: left inverted repeat
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- TAGAGTGAACCCCAAATTCCGGACTTTTT29
- (2) INFORMATION FOR SEQ ID NO:5:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 29 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: right inverted repeat
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- AAAAAGTCCAATTTTTGGGGTTCATACCA 29
- (2) INFORMATION FOR SEQ ID NO:6:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 429 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-1
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- ATGCAATTTAAATTTAAAAAAGTAAAAAGAAACAAATGAAATAGAGATATAAAAGGTTAT60
- TTAAAATTAAAACTTGATCAAAAGATAAAAATTATCGAGTTATATTTTCAAGAATTTAGT
120
- ATTTTAGAAATATCTAAAATAATGGAAAACTCTTATTCAGCATGCTATTCAGTAATAGAA180
- AAATACAAAAAGTTTGGTTATAATTCTTTTGCTATGGAAAAGAAAAAAGGAAGAAAATCT240
- AAAATAAATTTAGATGCTCAAAAGGCAACAAATTTTA AAATCAATATTGAAAATAAAATA300
- GAAAATAAAGATTTATTAATTAAACAATTAAAGGAAGAAAATAAAATACTCAAATTGGAG360
- AATGCGATAGCAAAAAAAGTGAGCGCCTTGGTTCAATTGAAAGACTCACTAACAAAGAAA420
- AATTCCAAA 429
- (2) INFORMATION FOR SEQ ID NO:7:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 309 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- ATGGTTGGTAAAATGTTAGAAAACGTAGAAGAGAATGGCCACAGCTTAAAAAATGTATTA60
- TTACATTCTGATCAAGGATGACAATACACTC ATCAAGATTATATTGATTATTTGAAAGAA120
- AAACAAACAACTCAAAGCATGTCAAGAAAGGGAAATTGTTTAGACAATAGTCCTACTGAA180
- TGTTTATTTAGTGTTATAAAAAGAGAATTTTGATTTGGAGAAGAAAAGAAATTTAATAGT240
- TTTAAAGAA TTTAAAACTGCTTTAGGAGATATATTTCATATTATAATAATGACAGAATTG300
- TTAATAAAT309
- (2) INFORMATION FOR SEQ ID NO:8:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 276 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-3
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- ATGAATATTAAAGCTTTTGGACTCCT ATTTATTTTTATGATAATTCTATTGATCATAACT60
- GTAATCGCTTATGCTTGACAAAATAAAGCGGCACCATCAATCACATATACAACTTTACTT120
- TGAGTTTTAATTTGTGTCTTTTCAATACTCACAATTCTTTCTCTTTATTTATTAATTTTA180
- TTT TTCATTGAATATGGTTTAATCAAAAAAATAGGTCTTAAAAAATCAGAACAAGAAATA240
- GAAGCATCTATAAGAAAATTTGTTAAATTTGCGATT276
- (2) INFORMATION FOR SEQ ID NO:9:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 143 amino acids
- (B) TYPE: amino acid
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-1
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- MetGlnPheLysPheLysLysValLysArgAsnLysTrpAsnArgA sp
- 151015
- IleLysGlyTyrLeuLysLeuLysLeuAsnGlnLysIleLysIleIle
- 202530
- GluLeuTyrPheGlnGluPheSerIleLeuGluIleSerLysIleMet
- 354045
- GluAsnSerTyrSerAlaCysTyrSerValIleGluLysTyrLysLys
- 505560
- PheGlyTyrAsnSerPheAlaMetGluLysLysLysGlyArgLysSer
- 65707580
- L ysIleAsnLeuAspAlaGlnLysAlaThrAsnPheLysIleAsnIle
- 859095
- GluAsnLysIleGluAsnLysAspLeuLeuIleLysGlnLeuLysGlu
- 100105110
- GluAsnLysIleLeuLysLeuGluAsnAlaIleAlaLysLysValSer
- 115120125
- Ala LeuValGlnLeuLysAspSerLeuThrLysLysAsnSerLys
- 130135140
- (2) INFORMATION FOR SEQ ID NO:10:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 103 amino acids
- (B) TYPE: amino acid
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- MetValGlyLysMetLeuGluAsnValGluGluAsnGlyHisSerLeu
- 15 1015
- LysAsnValLeuLeuHisSerAspGlnGlyTrpGlnTyrThrHisGln
- 202530
- AspTyrIleLysTyrLeuLys GluLysGlnThrThrGlnSerMetSer
- 354045
-
-
- ArgLysGlyAsnCysLeuAspAsnSerProThrGluCysLeuPheSer
- 5055 60
- ValIleLysArgGluPheTrpPheGlyGluGluLysLysPheAsnSer
- 65707580
- PheLysGluPheLysThrAlaLeuGly AspIlePheHisIleIleIle
- 859095
- MetThrGluLeuLeuIleAsn
- 100
- (2) INFORMATION FOR SEQ ID NO:11:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 92 amino acids
- (B) TYPE: amino acid
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: ORF-3
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- MetAsnIleLysAlaPheGlyLeuLeuPheIlePheMetIle IleLeu
- 151015
- LeuIleIleThrValIleAlaTyrAlaTrpGlnAsnLysAlaAlaPro
- 2025 30
- SerIleThrTyrThrThrLeuLeuTrpValLeuIleCysValPheSer
- 354045
- IleLeuThrIleLeuSerLeuTyrLeuLeuIleLeuPhePheIle Gly
- 505560
- TyrGlyLeuIleLysLysIleGlyLeuLysLysSerGlyGlnGlyIle
- 65707580
- GlyAlaSerIleArgLysPheValLysPheAlaIle
- 8590
- (2) INFORMATION FOR SEQ ID NO:12:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 57 amino acids
- (B) TYPE: amino acid
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (v ) FRAGMENT TYPE: internal
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Escherichia coli
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: IS3
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- AsnValIleValHisThrAspArgGlyGlyGlnTyrCysSerAlaAsp
- 1510 15
- TyrGlnAlaGlnLeuLysArgHisAsnLeuArgGlySerMetSerAla
- 202530
- LysGlyCysCysTyrAspAsnAlaCysValGlu SerPhePheHisSer
- 354045
- LeuLysValGluCysIleHisGlyGlu
- 5055
- (2) INFORMATION FOR SEQ ID NO:13:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 22 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RS 47 Primer
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
- GAATTCTTTA ATTGAGTTGCTC22
- (2) INFORMATION FOR SEQ ID NO:14:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 23 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RS 49 Primer
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
- TCCAAAAAGTCCGGAATTTGGGG23
- (2) INFORMATION FOR SEQ ID NO:15:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 24 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Myccoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RW004 Primer
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
- GGACTATTGTCTAAACAATTTCCC24
- (2) INFORMATION FOR SEQ ID NO:16:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 24 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RW005 Primer
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
- GGTTATTCGATTTCTAAATCGCCT24
- (2) INFORMATION FOR SEQ ID NO:17:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 24 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Mycoplasma fermentans
- (B) STRAIN: incognitus
- (vii) IMMEDIATE SOURCE:
- (B) CLONE: RW006 Probe
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
- GCTGTGGCCATTCTCTTCTACGTT24
-
-
-
- MainPage
http://www.rense.com
-
-
-
- This
Site Served by TheHostPros
|