U.S. patent application number 09/178098 was filed with the patent office on 2001-12-06 for method of identification of differentially expressed mrna.
Invention is credited to ALLAND, DAVID, BLOOM, BARRY R., KRAMNIK, IGOR.
Application Number | 20010049094 09/178098 |
Document ID | / |
Family ID | 22651181 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049094 |
Kind Code |
A1 |
ALLAND, DAVID ; et
al. |
December 6, 2001 |
METHOD OF IDENTIFICATION OF DIFFERENTIALLY EXPRESSED MRNA
Abstract
The method provided by the present invention sets forth a novel
combination of methods and principles which allows for the rapid
and accurate isolation and identification of a large number of
differentially expressed mRNAs.
Inventors: |
ALLAND, DAVID; (DOBBS FERRY,
NY) ; BLOOM, BARRY R.; (CAMBRIDGE, MA) ;
KRAMNIK, IGOR; (BRONX, NY) |
Correspondence
Address: |
CRAIG J ARNOLD
AMSTER ROTHSTEIN & EBENSTEIN
90 PARK AVENUE
NEW YORK
NY
10016
|
Family ID: |
22651181 |
Appl. No.: |
09/178098 |
Filed: |
October 23, 1998 |
Current U.S.
Class: |
435/6.12 ;
435/91.1; 435/91.2; 536/23.1 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12N 15/1072 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.2; 536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 019/34 |
Claims
What is claimed is:
1. A method of making a customized nucleic acid library comprising
nucleic acid sequences of interest and devoid of unwanted nucleic
acids, said method comprising the steps of: (a) obtaining a nucleic
acid library containing the nucleic acid sequences of interest and
unwanted nucleic acids; (b) screening said library for unwanted
nucleic acids; and (c) removing said unwanted nucleic acids from
said library resulting in a customized nucleic acid library
comprising nucleic acid sequences of interest and devoid of
unwanted nucleic acids.
2. The method of claim 1, wherein the customized nucleic acid
library comprises cDNA, RNA or genomic DNA.
3. The method of claim 2, wherein the cDNA, RNA or genomic DNA is
obtained from bacteria.
4. The method of claim 1, wherein the nucleic acid sequences of
interest are coding sequences.
5. The method of claim 1, wherein the unwanted nucleic acids are
selected from the group consisting of ribosomal RNA and other
nucleic acids that occur with high frequency in cellular RNA.
6. The method of claim 1, wherein the library is screened for
unwanted nucleic acids by contacting the library with nucleic acid
probes complementary to the unwanted nucleic acids.
7. The method of claim 6, wherein the nucleic acid probes are
labeled with detectable markers which permits detection of unwanted
nucleic acids upon hybridization with said probes.
8. A method of making a customized nucleic acid amplification
library comprising the steps of: (a) obtaining a nucleic acid
library containing the nucleic acid sequences of interest and
unwanted nucleic acids; (b) screening said library for unwanted
nucleic acids; (c) removing said unwanted nucleic acids from said
library resulting in a customized nucleic acid library comprising
nucleic acid sequences of interest and devoid of unwanted nucleic
acids; (d) excising the nucleic acid sequences of interest from the
customized nucleic acid library; and (e) purifying the nucleic acid
sequences of interest so excised to obtain a customized nucleic
acid amplification library.
9. The method of claim 1, wherein the customized nucleic acid
library comprises cDNA, RNA or genomic DNA.
10. The method of claim 8, wherein the cDNA, RNA or genomic DNA is
obtained from bacteria.
11. The method of claim 8, wherein the nucleic acid sequences of
interest are coding sequences.
12. The method of claim 8, wherein the unwanted nucleic acids are
selected from the group consisting of ribosomal RNA and other
nucleic acids that occur with high frequency in cellular RNA.
13. The method of claim 8, wherein the library is screened for
unwanted nucleic acids by contacting the library with nucleic acid
probes complementary to the unwanted nucleic acids.
14. The method of claim 13, wherein the nucleic acid probes are
labeled with detectable markers which permits detection of unwanted
nucleic acids upon hybridization with said probes.
15. The method of claim 8, wherein the nucleic acid sequences of
interest are purified to a size between about 400 to about 1500
base pair fragments.
16. A method of making a customized nucleic acid library comprising
the steps of: (a) obtaining a nucleic acid library containing
nucleic acid sequences of interest and unwanted nucleic acids; (b)
amplifying the nucleic acid sequences of interest in the library to
generate amplicons; and (c) pooling amplicons or subsets thereof
thereby obtaining a nucleic acid library containing nucleic acid
sequences of interest devoid of unwanted nucleic acids.
17. The method of claim 16 wherein the amplicons of step (b) are
immobilized on a solid support.
18. The method of claim 19, wherein the solid support is selected
from the group consisting of cellulose, nitrocellulose,
polystyrene, polypropylene, polysulfone, polyvinylidene fluoride
and polyethersulfone.
19. A method of detecting a nucleic acid sequence of interest in a
sample containing nucleic acid comprising the steps of: (a)
labeling the nucleic acid from the sample with a detectable marker;
(b) contacting the nucleic acid so labeled with the customized
nucleic acid amplification library produced by the method of claim
8 or 16 under conditions permitting the nucleic acid so labeled to
hydridize with the customized nucleic acid amplification library;
and (c) detecting hybridization of the labeled nucleic acid with
the customized nucleic acid amplification library.
20. A method of isolating a nucleic acid sequence of interest from
a sample containing nucleic acid comprising the steps of: (a)
labeling the nucleic acid from the sample with a detectable marker;
(b) contacting the nucleic acid so labeled with the customized
nucleic acid amplification library produced by the method of claim
8 or claim 16 under conditions permitting the nucleic acid so
labeled to hydridize with the customized nucleic acid amplification
library; and (c) isolating the hybridized nucleic acid so
detected.
21. A customized nucleic acid library comprising nucleic acid
sequences of interest and devoid of unwanted nucleic acids.
22. The library of claim 21, wherein the customized nucleic acid
library comprises cDNA, RNA or genomic DNA.
23. The library of claim 22, wherein the cDNA, RNA or genomic DNA
is obtained from bacteria.
24. The library of claim 21, wherein the nucleic acid sequences of
interest are coding sequences.
25. The library of claim 21, wherein the unwanted nucleic acids are
selected from the group consisting of ribosomal RNA and other
nucleic acids that occur with high frequency in cellular RNA.
26. The library of claim 21, which is contained in a vector.
27. The library of claim 21, wherein the nucleic acid sequences of
interest are amplified to form amplicons.
28. The library of claim 27, wherein the amplicons are immobilized
on a solid support.
29. The library of claim 28, wherein the solid support is selected
from the group consisting of cellulose, nitrocellulose,
polystyrene, polypropylene, polysulfone, polyvinylidene fluoride,
and polyethersulfone.
30. The library of claim 21, wherein the nucleic acid sequences of
interest are between about 400 to about 1500 base pairs in length.
Description
BACKGROUND OF THE INVENTION
[0001] The analysis of bacterial responses to environmental stimuli
can provide valuable insights into cellular mechanisms (1-5). This
approach is particularly well suited for studies of Mycobacterium
tuberculosis, a pathogen that must adapt to a variety of hostile
milieu including phagocytosis by macrophages and treatment with
antibiotics. Differential gene expression in bacteria has been
difficult to study because the absence of poly(A).sup.+ RNA
complicates removal of abundant ribosomal rRNA from low-abundance
mRNA. The number of differentially expressed genes that have been
identified in bacteria has been limited (6-11), except under
circumstances where large amounts of RNA can be obtained (12). It
recently has become possible to monitor gene expression in multiple
bacterial genes simultaneously by direct hybridization of total RNA
to high-density DNA arrays (12). However, the large amounts of
labeled RNA that must be hybridized to such arrays currently
restricts their utility in many biologically relevant
investigations. This problem is not resolved by amplification of
samples with the PCR because it often is not possible to amplify
complex mixtures of mRNA sequences while at the same time
maintaining their relative proportions (13). Accordingly, an
efficient and rapid method of identifying differentially expressed
mRNA would aid tremendously in understanding gene differential gene
expression.
SUMMARY OF THE INVENTION
[0002] The existing need for an efficient and rapid method of
identifying differentially expressed mRNA is met by the method
provided by the present invention. The method provided by the
present invention sets forth a novel combination of methods and
principles which allows for the rapid and accurate isolation and
identification of a large number of differentially expressed
mRNAs.
[0003] Additional objects of the invention will be apparent from
the description which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0004] FIGS. 1A-1C: Schematic representation of differential
expression using customized amplification libraries (DECAL). FIG.
1A shows the generation of customized Amplification libraries
(CAL). A cosmid library is screened for clones that contain
ribosomal DNA sequences. Non-ribosomal cosmids are digested into
similar sized fragments, gel purified, ligated to PCR adapters, and
PCR amplified. FIG. 1B shows positive selection and hybridization.
Reverse transcribed RNA samples are hybridized to a ribosomal DNA
free CAL, washed, then amplified to generate PCR probes. FIG. 1C:
The probes are labeled and hybridized to replicate colony arrays of
genomic plasmid libraries. Colonies that hybridize with differing
intensities to two PCR probes are selected for evaluation of
differentially expressed sequences.
[0005] FIGS. 2A and 2B: FIGS. 2A and 2B set forth the results of
hybridization of PCR probes to genomic DNA and plasmid digests.
DECAL was performed using RNA extracted from M. tuberculosis H37Rv
cultures that were either untreated (INH-), or treated with
isoniazid 1.0 .mu.g/ml for 18 hours (INH+). FIG. 2A sets forth
radiolabeled INH- cDNA (before positive selection with CAL), and
radiolabled INH- and INH+PCR probes (after positive selection with
CAL and amplification) were hybridized to H37Rv genomic digests.
The cDNA hybridized almost exclusively with a single band of
ribosomal DNA. The INH-PCR probe and INH+PCR probe both hybridized
to multiple sequences in the M. tuberculosischromosomal digests,
but showed no hybridization to the ribosomal band. FIG. 2B sets
forth Southern blots of M. tuberculosis H37Rv genomic DNA digested
with vuII, and PstI digests of six plasmids (P1-P6) that hybridized
differentially to the CR probes on colony array screening. Southern
blots were hybridized with adiolabeled INH-PCR probe (top), or
INH+PCR probe (bottom). The INH-PCR probe hybridized exclusively to
P6. The INH+PCR probe almost exclusively to P1 and preferentially
to P2 and P3. P4 and P5 did not hybridize differently to the two
probes and are unlikely to code for isoniazid induced genes.
[0006] FIG. 3: FIG. 3 shows the results of Induction of iniA after
treatment with different antibiotics. Autoradiographs of a Northern
blot containing RNA from M. tuberculosis cultures treated either
with no antibiotics; isoniazid 0.01 .mu.g/ml; isoniazid 0.1
.mu.g/ml; isoniazid 1 .mu.g/ml; ethambutol 5 .mu.g/ml; streptomycin
5 .mu.g/ml; and rifampin 5 .mu.g/ml. The blots were hybridized
first with an iniA DNA probe (top) to examine iniA induction; the
blot was then stripped and re-hybridized with a 16S probe (bottom)
to confirm equal RNA loading.
[0007] FIGS. 4A and 4B: FIGS. 4A and 4B set forth the results of
reverse transcription PCR of differentially expressed genes. FIG.
4A sets forth RNA was extracted from log phase M. tuberculosis
strain Erdman either without (lanes 1-3) or with (4-6) isoniazid
added to the bacterial cultures for the last 18 hours. RNA from
both cultures was equalized by comparison of the 23S band
intensity. RT PCR using three ten-fold dilutions of each RNA and
either iniA, asd or 16S specific primers was performed. Induction
of iniA and suppression of asd by isoniazid is demonstrated. The
amount of 16S RT PCR product is similar for equivalent dilutions,
indicating equal amounts of starting RNA. Lanes 7-8 are minus RT
controls; and lane 9 a negative PCR control. FIG. 4B sets forth
lack of iniA induction in an isoniazid resistant strain. Cultures
of isogenic BCG strain ATCC35735 which is susceptible to isoniazid
(lanes 1-6), or ATCC35747 which is resistant to isoniazid (lanes
7-12), were incubated either in the presence or absence of
isoniazid for the last 18 hours. Three ten-fold dilutions of RNA
extracted from each culture were tested by RT PCR for iniA
induction. Induction is seen only in the INH susceptible strain.
Lanes 13-16 are minus RT controls; and lane 17 a negative PCR
control containing no added template.
[0008] FIG. 5: FIG. 5 shows the limits to distinguishing
differences between samples. Ten-fold decreasing amounts of in
vitro transcribed M. tuberculosis inhA mRNA (tube 1 contained
1.multidot.10.sup.11 molecules; tube 2, 1.multidot.10.sup.10
molecules; tube 3, 1.multidot.10.sup.9 molecules; tube 4,
1.multidot.10.sup.8 molecules; tube 5, 1.multidot.10.sup.7
molecules; tube 6, no added molecules), and four-fold increasing
amounts of ask/asd mRNA (tube 1 contained no added molecules; tube
2, 4.multidot.10.sup.6 molecules; tube 3, 1.7.multidot.10.sup.7
molecules; tube 4, 6.multidot.10.sup.7 molecules; tube 5,
2.5.multidot.10.sup.8 molecules; tube 6, 1-10.sup.9 molecules),
were added to six tubes. Each tube also contained one microgram of
BCG total RNA. DECAL was performed separately for each tube. The
PCR probes were then hybridized to six Southern blots containing
ask/asd DNA, inhA DNA, and M. tuberculosis H37Rv genomic digests.
Autoradiography exposure was equalized to the hybridization
intensity of the H37Rv bands.
[0009] FIG. 6: Applying DECAL to small amounts of starting
material. Ten-fold decreasing amounts (1.multidot.10.sup.9 and
1.multidot.10.sup.8 molecules) of inhA mRNA, and four-fold
increasing amounts (1.multidot.10.sup.8 and 4.multidot.10.sup.8
molecules) of ask/asd mRNA were added to two tubes each containing
one microgram of BCG total RNA. The tubes were reverse transcribed
with biotin random primers, and serial ten-fold dilutions of the
cDNA (equivalent to 1 .mu.g, 100 ng and 10 ng of starting RNA) were
subjected to the DECAL method. The resulting PCR probes were
hybridized to duplicate Southern blots of a genomic M. tuberculosis
H37Rv digest, inhA DNA, and ask/asd DNA, to assess for the presence
of detectable differences in inhA and ask/asd signal.
Autoradiography exposure was equalized to the hybridization
intensity of the H37Rv bands.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention sets forth a novel approach for
studying differences in mRNA expression, which the inventors have
termed "differential expression using customized amplification
libraries" (DECAL), that permits global comparisons of bacterial
gene expression under varied growth conditions without a specific
requirement for DNA arrays. The key feature of DECAL technology is
the ability to amplify by PCR a complex mixture of expressed genes
in a reproducible and representative manner without the confounding
effects of rRNA or any other highly expressed gene product. The
inventors have found that three steps are essential for this
process: (i) removal of abundant sequences--in this case rRNA
sequences; (ii) reduction in the complexity of the sequences and
conversion of all cDNA sequences into fragments of similar size;
and (iii) selecting sequences that amplify efficiently. DECAL
accomplishes this by creating a customized amplification library
(CAL) of genomic sequences that has been manipulated for optimal
performance during PCR amplification. Instead of amplifying total
cDNA sequences, cDNA is hybridized to an excess of CAL,
nonhybridizing CAL sequences are removed and the remaining CAL
sequences are amplified without altering their proportion
representation. The amplified products derived from RNA samples can
be hybridized to replicate colony blots or colony arrays, and the
resulting hybridization patterns compare to determine the
differentially expressed genes present in the original RNA samples.
The inventors have herein demonstrated the applicability of the
DECAL system to the study of M. tuberculosis gene expression in
response to the antibiotic, isoniazid.
[0011] The present invention provides a method of making a
customized nucleic acid library comprising nucleic acid sequences
of interest and devoid of unwanted nucleic acids, said method
comprising the steps of: (a) obtaining nucleic acid library
containing the nucleic acid sequences of interest and unwanted
nucleic acids; (b) screening said library for unwanted nucleic
acids; and (c) removing said unwanted nucleic acids from said
library resulting in a customized nucleic acid library comprising
nucleic acid sequences of interest and devoid of unwanted nucleic
acids. The customized nucleic acid library may comprise cDNA, RNA
or genomic DNA. The cDNA, RNA or genomic DNA may be obtained from
bacteria. In a preferred embodiment of the invention, the cDNA, RNA
or genomic DNA is obtained from mycobacteria. The nucleic acid
sequences of interest may be, for example, coding sequences,
sequences corresponding to a particular class of genes and
sequences of a particular family of genes. The unwanted nucleic
acids are predetermined depending on the genes of interest. If a
library containing the coding sequences of an entire genome is
desired, the unwanted nucleic acids would be, for example,
ribosomal RNA and other nucleic acids that occur with high
frequency in cellular RNA. The library is screened for unwanted
nucleic acids by contacting the library with nucleic acid probes
complementary to the unwanted nucleic acids. The nucleic acid
probes may be labeled with detectable markers which permits
detection of unwanted nucleic acids upon hybridization with said
probes. Non-limiting examples of detectable markers include
fluorescence, enzymes, and radiolabeled markers, such as
radiolabeled isotopes and biotin.
[0012] Further provided by the present invention is a method of
making a customized nucleic acid amplification library comprising
the steps of: (a) obtaining a nucleic acid library containing the
nucleic acid sequences of interest and unwanted nucleic acids; (b)
screening said library for unwanted nucleic acids; (c) removing
said unwanted nucleic acids from said library resulting in a
customized nucleic acid library comprising nucleic acid sequences
of interest and devoid of unwanted nucleic acids; (d) excising the
nucleic acid sequences of interest from the customized nucleic acid
library; and (e) purifying the nucleic acid sequences of interest
so excised to obtain a customized nucleic acid amplification
library. The customized nucleic acid library may comprise cDNA, RNA
or genomic DNA. The cDNA, RNA or genomic DNA may be obtained from
bacteria. In a preferred embodiment of the invention, the cDNA, RNA
or genomic DNA is obtained from mycobacteria. The nucleic acid
sequences of interest may be, for example, coding sequences,
sequences corresponding to a particular class of genes and
sequences of a particular family of genes. The unwanted nucleic
acids are predetermined depending on the genes of interest. If a
library containing the coding sequences of an entire genome is
desired, the unwanted nucleic acids would be, for example,
ribosomal RNA and other highly expressed gene products. The library
is screened for unwanted nucleic acids by contacting the library
with nucleic acid probes complementary to the unwanted nucleic
acids. The nucleic acid probes may be labeled with detectable
markers which permits detection of unwanted nucleic acids upon
hybridization with said probes. Non-limiting examples of detectable
markers include fluorescence, enzymes, and radiolabeled markers,
such as radiolabeled isotopes and biotin. The nucleic acid
sequences of interest are purified to a particular size in order to
generate a library of similarly sized fragments. In a particular
embodiment of the invention, the nucleic acid sequences of interest
are purified to a size between about 400 to about 1500 base pair
fragments, although it is to be understood that other sizes of
nulcleic acids may be purified.
[0013] The present invention also provides a method of making a
customized nucleic acid library comprising the steps of: (a)
obtaining a nucleic acid library containing nucleic acid sequences
of interest and unwanted nucleic acids; (b) amplifying the nucleic
acid sequences of interest in the library to generate amplicons;
and pooling amplicons or subsets thereof thereby obtaining a
nucleic acid library containing nucleic acid sequences of interest
devoid of unwanted nucleic acids. The separate, unpooled amplicons
may be immobilized on a solid support. The solid support may be
selected from the group consisting of, for example, cellulose,
nitrocellulose, polystyrene, polypropylene, polysulfone,
polyvinylidene fluoride, and polyethersulfone.
[0014] Further provided by the present invention is a method of
detecting a nucleic acid sequence of interest in a sample
containing nucleic acid comprising the steps of: (a) labeling the
nucleic acid from the sample with a detectable marker; (b)
contacting the nucleic acid so labeled with the customized nucleic
acid amplification library produced by the methods described above
under conditions permitting the nucleic acid so labeled to
hybridize with the customized nucleic acid amplification library;
and (c) detecting hybridization of the labeled nucleic acid with
the customized nucleic acid amplification library.
[0015] Also provided by the present invention is a method of
isolating a nucleic acid sequence of interest from a sample
containing nucleic acid comprising the steps of: (a) labeling the
nucleic acid from the sample with a detectable marker; (b)
contacting the nucleic acid so labeled with the customized nucleic
acid amplification library produced by the method described above
under conditions permitting the nucleic acid so labeled to
hybridize with the customized nucleic acid amplification library;
(c) detecting hybridization of the labeled nucleic acid with the
customized nucleic acid amplification library; and (d) isolating
the hybridized nucleic acid so detected.
[0016] The present invention further provides a customized nucleic
acid library comprising nucleic acid sequences of interest and
devoid of unwanted nucleic acids. The customized nucleic acid
library may comprise cDNA, RNA or genomic DNA. The cDNA, RNA or
genomic DNA may be obtained from bacteria. In a preferred
embodiment of the invention, the cDNA, RNA or genomic DNA is
obtained from mycobacteria. The nucleic acid sequences of interest
may be, for example, coding sequences, sequences corresponding to a
particular class of genes and sequences of a particular family of
genes. The library may contain the nucleic acid sequences of
interest in vectors, such as cosmids or plasmids. The nucleic acid
sequences of interest are amplified to form amplicons, which are
then immobilized on a solid support. The solid support may be
cellulose, nitrocellulose, polystyrene, polypropylene, polysulfone,
polyvinylidene fluoride, or polyethersulfone. In a particular
embodiment of the invention, the nucleic acid sequences of interest
are purified to a size between about 400 to about 1500 base pair
fragments, although it is to be understood that other sizes of
nulcleic acids may be purified.
[0017] Despite the many advantages of the DECAL technique, the use
of colony arrays to detect PCR probe hybridization limits the
ability of DECAL to perform truly global gene expression screens.
Medium sized plasmid inserts usually contain sequences
complementary to several open reading frames. This can result in
decreased sensitivity for detecting differential mRNA expression
when one gene on the plasmid is induced, but others on the same
plasmid are repressed. Furthermore, it is laborious to evaluate
every open reading frames on a differentially expressed plasmid in
order to identify the actual differentially expressed gene.
[0018] The development of DNA array (chip) technology offers an
elegant solution to this problem. DECAL can enhance the sensitivity
of DNA array-based detection methods by providing probes that can
be PCR amplified without significantly altering mRNA
representation. DECAL generated PCR probes can be fluorescently
labeled and hybridized to micro arrays containing short PCR
amplicons from every M. tuberculosis open reading frame. The
resulting fluorescent pattern will permit a clear determination of
which CAL sequences represent differentially expressed genes. Using
this approach, DECAL should extend the applicability of DNA arrays
to investigations where limited amounts of initial RNA is
available.
[0019] Adapt DECAL to Enable Hybridization with DNA Microarrays
[0020] CAL sequences are unlikely to regularly cross hybridize to
the probes that have been placed on DNA arrays. To ensure 100%
cross hybridization with DNA microarrays, and to enable DNA arrays
to "read" the results of subsequent DECAL experiments, we will
create CALs using amplicons identical to those used in DNA array
construction.
[0021] Determine the Optimal CAL Complexity, and Create CALs
Containing more Limited Sequences for Ultra Sensitive Screens
[0022] It is possible optimal proportional amplification will
require CALs with reduced complexity compared to CALs constructed
with the entire set of M. tuberculosis open reading frame
amplicons. This will be investigated using CALs derived from
amplicon subsets. In order to perform ultra sensitive differential
expression screens, we will take advantage of the completely
sequenced M. tuberculosis genome to design CALs with sequences
limited to genes relevant to a specific area of investigation.
[0023] Develop DECAL for Use with Limiting Amounts of Bacterial RNA
that is Mixed with Contaminating Human/host Sequences
[0024] DECAL can be performed with nanogram quantities of starting
RNA; however, conditions have not been optimized for investigations
where large amounts of contaminating foreign DNA or RNA is present.
In experiments using M. tuberculosis RNA spiked into human and
mouse RNA, we will determine the conditions that permit DECAL to be
performed in the presence of large amounts of non-hybridizing
nucleic acid sequences. One goal of this aim will be to determine
the minimal amount of manipulations necessary to obtain
sufficiently pure bacterial RNA for DECAL experiments.
[0025] Study Gene Expression in Human Sputum before and during
Early Treatment
[0026] Gene expression will be investigated in order to determine
the early changes that predict response to treatment. Microbial
factors induced by the host immune response will also be
investigated as potential vaccine candidates. This aim follows from
the successful completion of aims 1-3. We will compare M.
tuberculosis gene expression during human infection, by performing
DECAL- DNA MICROARRAY assays of RNA isolated from sputum samples.
Gene expression will be investigated with different stages and
types of disease, different antibiotic treatments, and in patients
with rapid and slow response to therapy.
[0027] Study Gene Expression in Laminal Models of Infection
[0028] We will study and compare gene expression in mammalian host
tissues in the early, middle and late stages of infection.
[0029] The present invention is described in the following
Experimental Details Sections which is set forth to aid in the
understanding of the invention, and should not be construed to
limit in any way the invention as defined in the claims which
follow thereafter.
Experimental Details Section
[0030] A. Materials and Methods
[0031] Libraries and Plasmids
[0032] Cosmid libraries were constructed by ligation of Sau3A
partial digests of M. tuberculosis H37Rv into pYUB328 (14). Plasmid
libraries were constructed by ligation of complete PstI or SacI
digests of M. tuberculosis H37Rv into pUC19 (15). The plasmid pUB
124 was constructed by insertion of a 1.7 kb PstI fragment of the
ask/asd operon containing the down stream portion of the M.
tuberculosis ask gene and the complete asd gene into pKSII (16).
The plasmid PET-inhA, containing an 800 base-pair fragment of the
M. tuberculosis inhA gene inserted into the BamHI site of pET-23a+
(Novagen, Madison Wis.) was a kind gift of Dr. John Blanchard
(Albert Einstein College of Medicine, Bronx N.Y.).
[0033] Creation of Ribosomal Free Customized Amplification
Libraries One thousand cosmid library clones were inoculated into
"master" 96 well microtiter plates containing L broth and
ampicillin 50 .mu.g/ml, transferred by a pronged "frog" onto
Biotrans nylon membranes (ICN Pharmaceuticals, CostaMesa, Calif.),
and hybridized separately with [.alpha..sup.32p] radiolabled
(Megaprime labeling kit, Amersham, Arlington Heights, Ill.) PCR
probes to M. tuberculosis ribosomal 5S, 16S, and 23S genes.
Fourteen cosmids containing ribosomal DNA were identified;
non-ribosomal cosmids were re-inoculated from master plates and
individually cultured. Cosmids were extracted by SDS/alkaline lysis
(17) in pools of 16. Cosmid DNA was pooled, digested with PacI,
which does not restrict the M. tuberculosis genome, and insert DNA
was purified from an agarose gel by electro-elution. Approximately
1 .mu.g of precipitated DNA was digested with AluI and 100 ng run
on a 2% NuSieve GTG low melting point agarose gel (FMC Bioproducts,
Rockland, Me.). Marker DNA was run simultaneously in a separate gel
to avoid cross contamination of samples. The gels were aligned, and
the section corresponding to 400-1,500 base pairs of the AluI
digest was excised. Five .mu.l of gel slice was ligated with 1
.mu.l of Uniamp XhoI adapters 2 pmol/).mu.l (Clonetech, Palo Alto,
Calif.) in 20 .mu.l total volume. Ten .mu.l of the ligation was PCR
amplified with 2 .mu.l of 10 .mu.M Uniamp primers (Clonetech),
1.times. vent polymerase buffer and 0.8 units of Vent (exo-)
polymerase (New England Biolabs, Beverly, Mass.) in 100 .mu.l total
volume. After a five minute hot start, ten cycles of PCR with one
minute segments of 95.degree. C., 65.degree. C., and 72.degree. C.,
were followed by the addition of 3.2 units of Vent (exo-)
polymerase and 27 additional cycles of 95.degree. C. for one
minute, 65.degree. C. for two minutes, and 72.degree. C. for three
minutes. Uniamp primer sequence: 5'-CCTCTGAAGGTTCCAGAATCGATAG-3';
Uniamp XhoI adapter sequence top strand:
5'-CCTCTGAAGGTTCCAGAATCGATAGCTCGAGT-3'; bottom strand:
5'-P-ACTCGAGCTATCGATTCTGGAACCTTCAGAGGTT7-3'.
[0034] RNA Extraction
[0035] Mycobacterial cultures were grown to mid Log phase in
Middlebrook 7H9 media supplemented with OADC, 0.05% Tween 80, and
cyclohexamide (18) (for some experiments antibiotics were added for
the last 18 hours), pelleted, resuspended in chloroform/methanol
3:1, and vortexed for 60 seconds or until the formation of an
interface. RNA was extracted with five volumes of Triazole (Life
Technologies, Gaithersburg, Md.), the aqueous layer precipitated in
isopropanol, redissolved in 4M GTC and extracted a second time with
Triazole.
[0036] Positive Selection
[0037] One .mu.g of RNA was reverse transcribed with 7.7 .mu.g
biotin labeled random hexamers and biotin DATP (one tenth total
dATP) using superscript II (Gibco BRL, Grand Island, N.Y.) at
50.degree. C. for one hour, RNAse H was then added for one half
hour at 37.degree. C. Three hundred ng of CAL, 20 .mu.g of salmon
sperm DNA, and 20 .mu.g of tRNA were added to the cDNA for a final
volume of 150 .mu.l. The sample was phenol/chloroform extracted
twice, ethanol precipitated overnight, resuspended in 6 .mu.l of 30
mM EPPS (Sigma), pH 8.0/3 mM EDTA, overlain with oil, and heated to
99.degree. C. for 5 minutes, then 1.5 .mu.l of 5 M NaCl preheated
to 69.degree. C. was quickly added (19). The sample was incubated
at 69.degree. C. for three to four days, then diluted with 150
.mu.l of incubation buffer (1.times.TE, 1 M NaCl, 0.5% Tween 20)
that had been preheated to 69.degree. C., and 50 .mu.l of washed,
preheated streptavadin coated magnetic beads (Dynal, Oslo, Norway)
were then added. The sample was then incubated at 55.degree. C.
with occasional mixing for 30 minutes, washed three times at room
temperature and three times 30 minutes at 69.degree. C. with 0.1%
SDS, 0.2.times.SSC by placing the microfuge tubes into a larger
hybridization tube in a rotating microhybridization oven (Bellco,
Vineland, N.J.). The sample was then washed with 2.5 mM EDTA and
eluted by boiling in 80 .mu.l of water. PCR was performed as in the
CAL preparation using 20 .mu.l of sample.
[0038] Colony Array Hybridizations
[0039] Genomic plasmid library arrays were prepared by Genome
Systems (St. Louis, Mo.) by robotically double spotting 9,216
colonies from the PstI and SacI plasmid libraries onto replicate
nylon membranes. PCR probes were labeled by random priming with
[.alpha..sup.32P] dCTP (Megaprime labeling kit, Amersham) for at
least 6 hours, hybridized to the colony arrays in Rapid-hyb buffer
(Amersham), washed at 69.degree. C. in 0.1.times.S.C., 0.1% SDS,
and visualized by autoradiography. Double spotted colonies which
hybridized at different intensities with two PCR probes were
selected for further analysis.
[0040] Northern Blots
[0041] Five .mu.g of each RNA sample were analyzed by northern blot
with Northern Max kits (Ambion, Austin, Tex.) in a 1% denaturing
agarose gel, probed with inserts of differentially expressed
plasmids labeled by random priming with [.alpha..sup.32P] dCTP, and
visualized by autoradiography.
[0042] Southern Blots
[0043] Plasmid or genomic DNA was digested with restriction
enzymes, subjected to electrophoresis in a 1% agarose gel and
transferred by capillary action to Biotrans nylon membranes. The
blots were hybridized and washed as in "colony array
hybridizations" above, and visualized by autoradiography.
[0044] Reverse Transcription PCR
[0045] One microgram of RNA was reverse transcribed using the
appropriate reverse PCR primer and superscript II at 50.degree. C.
For iniA and asd, three serial ten-fold dilutions of cDNA were
made; 16S cDNA was diluted 1 in 10.sup.6, 1 in 10.sup.7, and 1 in
10.sup.8. PCR was performed with Taq polymerase and 1.times.PCR
buffer (Gibco BRL) containing 2 mM MgCl.sub.2 for 25 cycles
annealing at 60.degree. C. for iniA; 35 cycles annealing at
58.degree. C. for asd; 25 cycles annealing at 63.degree. C. for
16S. PCR products were analyzed on a 1.7% agarose gel, images were
stored to disk by digital camera (Appligene, Pleasanton, Calif.),
and the amounts of PCR product were calculated by densitometry
(Imaging Software, National Institute of Health, Bethesda, Md.).
Primers used for iniA: 5'-GCGCTGGCGGGAGATCGTCAATG-3',
5'-TGCGCAGTCGGGTCACAGGAGTCG-3'; for asd: 5'-TCCCGCCGCCGAACACCTA-3',
5'-GGATCCGGCCGACCAGAGA-3'; for 16S: 5'-GGAGTACGGCCGCAAGGCTAAAAC-3',
5'-CAGACCCCGATCCGAACTGAGACC-3'.
[0046] In Vitro Synthesis of InhA and ask/asd mRNA
[0047] Plasmid vectors PET-inhA (inhA mRNA synthesis) and pUB124
(ask/asd mRNA synthesis) were digested with HindIII and BstXI
respectively to terminate transcription immediately downstream of
the transcribed genes. Transcription was performed for 1 hour at
37.degree. C. in 1.times. transcription buffer (Promega, Madison,
Wis.) containing 500 ng of restricted plasmid DNA, 0.4 mM NTP's, 40
units RNAsin (Promega), and either 60 units of T7 RNA polymerase
(Promega) for PET-inhA, or 60 units of T3 RNA polymerase (Promega)
for pUB124. One unit of DNAse (DNAse R1Q, Promega) was then added
to each tube and the reaction incubated for an additional 30
minutes. RNA was purified after DNAse treatment using RNeasy
columns (Qiagen, Santa Clarita, Calif.), and quantitated by
spectrophotometry. Complete plasmid DNAse treatment and mRNA
synthesis was confirmed on both non-denaturing and denaturing
agarose gels.
[0048] Adapt DECAL for Detection with DNA Arrays
[0049] CAL sequences are derived from size fractionated. Alul
digests of M. tuberculosis genomic DNA. In contrasts, the probes
present on DNA arrays are selected by computer for their uniqueness
and their ability to be efficiently amplified by PCR. The CAL and
DNA array sequences are unlikely to consistently cross hybridize.
We will work in collaboration with investigators at Stanford
University to resolve this problem. The goal will be to construct a
CAL that can fully hybridize to DNA arrays. The amplicon sequences
used in DNA array construction will be pooled and ligated with
adapters to construct CALs. Currently, a single adapter sequence is
used at both ends of the amplicons, but additional sensitivity may
be obtained when two different adapter sequences are used. This
type of CAL can be prepared by the simultaneous ligation of two
adapter sequences followed by PCR with both complementary primers.
Under the appropriate conditions, sequences will be preferentially
amplified (Diatchenko et al., 1996). Initial experiments have been
successful at making a single adapter CAL using amplicons pooled
from 80% of the M. tuberculosis open reading frames. The
representation of this CAL will be assessed by hybridization to the
DNA arrays at Stanford University. Subsequent modifications in CAL
preparation will be based on the results of the CAL-DNA array
hybridization experiments. Improvements in CAL representation will
be achieved by modifying adapter sequences, and adjusting the
ligation and PCR conditions.
[0050] Determine the Optimal CAL Complexity, and Create CALs
Containing more Limited Sequences for Ultra Sensitive Screens.
[0051] Proportional amplification of the approximately 4000 pooled
DNA array amplicons may not be achievable in a single CAL
preparation. Simultaneous amplification of this large number of
sequences may be difficult to achieve even under the conditions of
DECAL. It is also possible that specific sequences may be
particularly resistant to amplification in a multiplex assay, or
may amplify preferentially. DECAL is uniquely able to resolve these
issues because the complexity of PCR amplification can be
controlled by using greater or lesser numbers of sequences in CAL
construction. A series of CALs will be created with increasing
numbers of pooled amplicons. The optimal CAL complexity will be
investigated in two ways. 1) For each CAL, the sample will be split
into four, and separate DECAL experiments will be performed on an
RNA sample that has been spiked with a number of different mRNA
transcripts. The four resulting PCR will be hybridized to Southern
blots containing the spiked sequences. The PCR probes will also be
hybridized to DNA arrays. Proportional amplification will be
assessed in a manner similar to the experiments outlined in
preliminary data (FIGS. 2 and 3), and the consistency of
amplification will be assessed by measuring the inter-assay
variability of the four parallel DECAL experiments as they
hybridize to the Southern blots and DNA arrays. 2) Large amounts of
fluorescent RNA from M. tuberculosis cultured under two in vitro
conditions (such as with and without isoniazid) will be hybridized
directly to DNA arrays. DECAL experiments will be performed on
these same RNA samples using CALs of increasing complexity. The
proportional amplification and overall representation of each of
the DECAL experiments will be assessed using the direct DNA array
hybridization as a "gold standard." This will also permit the
identification of "problem sequences" that are resistant to
proportional amplification. These sequences will be omitted from
subsequent CALs and will be incorporated into a separate low
complexity CAL. If necessary, a number of reduced complexity CALs
will be used in combination for subsequent studies.
[0052] It is not necessary to investigate the entire complement of
M. tuberculosis expressed genes in every study. For example,
studies of the in vivo bacterial response to isoniazid could
initially be restricted to a subset (albeit large group) of genes
involved in fatty acid biosynthesis. Other genes identified as
regulated by isoniazid in vitro could also be included. Lower
complexity CALs will be constructed that contain subsets of M.
tuberculosis open reading frame amplicons specifically chosen for
these types of studies. Because these CALs will contain many less
sequences, each sequence should be amplified to a greater extent,
permitting accurate investigations starting with very small amounts
of material. These CALs will be carefully evaluated for overall
sensitivity and roportional amplification as outlined above.
[0053] Develop DECAL for use with Limiting Amounts of Bacterial RNA
hat is Mixed with Contaminating Human/host Sequences.
[0054] Preliminary studies have demonstrated that DECAL can be
performed with nanogram quantities of starting RNA. However DECAL
has not been optimized for samples containing excess amounts of
contaminating sequences will effect DECAL performance as the
current assay is successful despite (because of) the inclusion 20
micrograms of RNA and salmon sperm DNA in the positive selection
step. However, samples obtained from solid organs may contain
milligram quantities of host RNA. Different protocols will be
investigated to determine the minimal amount of manipulations
necessary to obtain sufficiently pure bacterial RNA for DECAL
experiments. First M. tuberculosis RNA will be added into human
RNA, and DECAL experiments will be performed as in aim 1 to
estimate the amount of permissible contamination. Next, M.
tuberculosis bacilli will be mixed with human and mouse samples,
and different protocols of RNA extraction will be assessed for
their impact on DECAL studies. In sputum, the benefit of DNAase and
RNAase treatment prior to M. tuberculosis cell lysis and RNA
extraction will be investigated. In mouse tissue, we will test the
following protocol: Spleen, liver and lungs will be homogenized in
mesh tubing containing PBS with 1% each of TrixtonX 100 and
deoxycholate to lese host tissue cells. This detergent treatment
has no effect on the viability of the bacilli. issue suspensions
will then be centrifuged after vigorous vortexing to obtain
bacterial pellets. Four hundred micro liters of chloroform/methanol
(3:1) will be added followed by three minutes of vortexing. Glass
beads and two milliliters of Trizol Reagent (Life Technology) will
then be added. The mixture will be vortexed for two minutes and 100
micro liters of CHCl.sub.3 will be added. RNA will then be
extracted as per the manufacturer's protocol and treated with
DNAase. The RNA will then be recovered using RNeasy columns
(Qiagen). This protocol has resulted in M. tuberculosis RNA of
sufficiently good quality for RT PCR assays (Chan J, personal
communication), and is likely to yield RNA that can be used in
DECAL experiments.
[0055] Study Gene Expression in Human Sputum before and During
Early Treatment.
[0056] Microbial factors expressed during various stages of M.
tuberculosis infection will be investigated using CALs composed of
DNA array amplicons. Experiments will be performed under the
conditions optimized in aims 1-3. Sputum samples will be obtained
from patients before and during treatment under TBRU study
protocols. Changes in gene expression will be investigated in both
patients undergoing mono therapy and in patients receiving multi
drug therapy. Both induction and suppression of genes will be
investigated in order to determine the early changes that predict
two month and four month culture negativity, and long term relapse
free cures.
[0057] The second goal of this aim will be to determine if there is
a relationship between bacterial gene expression and the type and
extent of pulmonary tuberculosis. The host immune response clearly
plays an important role in tuberculosis. However, primary bacterial
factors may also influence disease progression, and these factors
may in turn be modulated by host immune responses. Genes with
increased expression during more advanced stages of disease may be
potential vaccine candidates. Gene expression in patients with
different stages of pulmonary infections that are enrolled in other
TBRU studies will be compared. Sputum from relatively asymptomatic
patients with pulmonary tuberculosis will be obtained as part of
the household contact study. Preserved samples from the initial
household case and other cases with more severe disease will also
be used for comparison HIV positive and HIV negative subjects with
similar stages of disease will also be examined.
[0058] Study Gene expression in Laminal Models of Infection
[0059] Animal models of infection will permit a more systematic
study of gene expression during specific stages of disease. These
investigations are important in that they may reveal new virulence
determinants and vaccine candidates. Mice will be infected with
H37rv both intravenously and via the aerosol route using the low
dose infection model. Lung, liver and spleen will be harvested
during early, middle and late stages of infection. The tissue will
be processed as described in aim 3 and DECAL experiments
performed.
[0060] B. Results
[0061] Creation of an M. tuberculosis customized amplification
library (CAL). A representative genomic library of the entire M.
tuberculosis genome was "customized" for proportional amplification
by PCR (FIG. 1A). A critical requirement for the amplification
library was that all DNA encoding rRNA genes had to be removed
completely so that these highly abundant sequences could not
confound proportional amplification in subsequent steps. This was
performed by screening an M. tuberculosis cosmid library for rRNA
gene sequences and removing all positive clones. The remaining rRNA
free M. tuberculosis genomic sequences were excised from their
cosmid vector and pooled. A second requirement was that the
amplification library contain DNA sequences of relatively uniform
size, and that their complexity be reduced in comparison to the
entire M. tuberculosis genome. This was accomplished by restricting
the excised inserts with AluI into small fragments and recovering
the sequences between 400 and 1,500 base pairs in length by gel
purification. The purified sequences were ligated to adapters for
subsequent PCR. The final requirement for the amplification library
was that all CAL sequences needed to be efficiently amplifiable by
PCR. The AluI enzyme typically restricts M. tuberculosis open
reading frame several times; we selected for the most efficiently
amplifiable fragments by subjecting the library to a number PCR
cycles prior to the subsequent hybridization steps.
[0062] Liquid Hybridization and Positive Selection
[0063] During positive selection, cDNA is hybridized to the CAL,
and CAL sequences that are non-complementary to the cDNA are
removed (FIG. 1B). In order to identify the M. tuberculosis genes
that were induced by isoniazid, M. tuberculosis was cultured to
mid-log phase; the culture was then split and grown an additional
18 hours either in the absence (INH-) or presence (INH+) of
isoniazid (1 .mu.g/ml). Total cellular RNA was then extracted
separately from both cultures, and reverse transcribed to biotin
labeled cDNA. The biotin-cDNA was used to capture complementary CAL
sequences on streptavidin beads. The beads were extensively washed,
and the remaining CAL sequences were re-expanded by PCR. We term
the products of this procedure "PCR probes". Despite the fact that
the biotin cDNA from each sample was primarily ribosomal, neither
INH- nor INH+PCR probes contained ribosomal sequences because no
amplifiable ribosomal DNA was present in the CAL. However, each PCR
probe hybridized to multiple non-ribosomal sequences when tested
against an M. tuberculosis PvuII genomic digest (FIG. 2A) or
assayed for hybridization to M. tuberculosis groES and hsp65 DNA,
and randomly selected M. tuberculosis cosmids (data not shown).
[0064] Detection and Evaluation of Differential Gene Expression
[0065] Differentially expressed genes were determined by examining
the differential hybridization patterns of the PCR probes (FIG.
1C). PCR probes derived from INH- and INH+RNA samples were
radiolabeled and hybridized to replica membranes containing arrays
of colonies from an M. tuberculosis genomic library. Hybridization
signals to most colonies were equal when small differences in
background were accounted for, but a subset of colonies was found
to hybridize more strongly to either the INH- or INH+ probe. Six
colonies were selected for further evaluation; five hybridized more
strongly with the INH+ probe (P1-P5) and one hybridized more
strongly with the INH- probe (P6). Differential hybridization was
confirmed for P1, P2, P3, and P6 by re-hybridizing the INH- and
INH+PCR probes to duplicate Southern blots of the excised plasmid
inserts (FIG. 2B). P1 and P6 hybridized almost exclusively to the
appropriate of the PCR probes, while P2 and P3 hybridized to both
probes but with different intensities. P4 and P5 were found not to
hybridize differentially on secondary screen. The ends of the
plasmid inserts were sequenced and aligned to the completely
sequenced M. tuberculosis genome (20). P1 and P2, which encoded
sequences that hybridized almost exclusively with the INH+ probe
were homologous to a set of predicted proteins. P1 encoded a
sequence identical to Rv0342, a large open reading frame that
appeared to be the second gene of a probable three gene operon.
This open reading frame was named iniA (isoniazid induced gene A),
and the upstream open reading frame Rv0341, was named iniB. P2
encoded a sequence that was not complementary to P1, but that was
identical to the third gene in the same probable operon Rv0343,
this open reading frame was named iniC. A putative protein encoded
by the iniA gene was found to contain a phosphopantetheine
attachment site motif (21) suggesting that it functions as an acyl
carrier protein. Both iniA and iniC lacked significant homology to
other known genes but were 34% identical to each other. A sequence
similarity search demonstrated that iniB had weak homology to
alanineglycine rich cell wall structural proteins (22). Northern
blot analysis using excised inserts to probe total RNA from M.
tuberculosis cultured in the presence or absence or different
antibiotics verified that iniA was strongly induced by isoniazid
and ethambutol, drugs that act by inhibiting cell wall biosynthesis
but not by rifampin or streptomycin, agents that do not act on the
cell wall (FIG. 3). P3, which also encoded a sequence that
preferentially hybridized to the INH+ probe contained a 5 kb insert
spanning M. tuberculosis cosmids MTCYH10 and MTCY21D4. This region
contained multiple small open reading frames, most with no known
function. Northern blot analysis using the 5 kb insert as a probe
confirmed that P3 preferentially hybridized to RNA from M.
tuberculosis that had been cultured in the presence of isoniazid
(data not shown). P6, which encoded a sequence hybridizing
predominantly with the INH- probe was found to encode
L-aspartic-Psemialdehyde dehydrogenase (asd). The asd gene is an
important component of the diaminopimelate pathway required for
biosynthesis of the peptidoglycan component of bacterial cell
walls. Modulation of asd by a cell wall antibiotic such as
isoniazid is not unexpected.
[0066] Reverse transcription (RT) PCR assays confirmed differential
gene expression of both asd and iniA (FIG. 4A), as well as of iniB
and iniC (data not shown). As predicted, iniA was strongly induced
by isoniazid (70 fold induction by densitometry), while asd was
repressed (17 fold). Induction of iniA was also tested in two
isogenic strains of BCG that were either sensitive or resistant to
isoniazid. The resistant phenotype was conferred by a mutation in
katG which normally converts isoniazid from a prodrug to its active
form (23). Induction of iniA was seen only in the susceptible BCG
strain demonstrating the requirement for isoniazid activation (FIG.
4B).
[0067] Detecting Limited Differences in Gene Expression and Rare
mRNAs
[0068] Most RNA subtraction techniques have a limited ability to
detect differentially expressed genes that are present in both
bacterial populations. We determined that the DECAL method can
distinguish small differences in gene expression, and can detect
rare mRNA sequences. Ten-fold dilutions of in vitro transcribed
mRNA from the M. tuberculosis inhA gene were added to six tubes
each containing one microgram of BCG total RNA (equivalent to
approximately 1.multidot.10.sup.7 bacilli). In vitro transcribed
mRNA from the M. tuberculosis ask/asd operon was added to the same
tubes in four-fold increasing amounts. The DECAL method was
performed separately on each tube, and the relative proportions of
amplified inhA and ask/asd CAL sequences were measured by
hybridization of each PCR probe to identical Southern blots (FIG.
5). Decreasing inhA signal is apparent from 1.multidot.10.sup.11 to
1.multidot.10.sup.7 transcripts (1:20 w/w to 1:200,000 w/w) when
normalized by equal hybridization to PvuII genomic digests of M.
tuberculosis strain H37Rv. Increases in ask/asd signal can be
detected beginning at 1.6.multidot.10.sup.7 transcripts (1:64,000
w/w), and the signal clearly increased with each four-fold increase
in added transcript. At lower amounts of added ask/asd or inhA
mRNA, the signal merged with the background from the BCG RNA
present in each tube. These results demonstrate that representative
and proportional amplification is maintained in six separate
samples, and confirm the ability of DECAL to detect small
differences in gene expression for both high and low abundance
mRNAs.
[0069] Differential gene expression in small quantities of RNA. To
investigate the sensitivity of the method, i.e. the minimum amount
of starting RNA required, decreasing amounts of inhA mRNA, and
increasing amounts of ask/asd mRNA were added to two tubes each
containing one microgram of BCG total RNA. The tubes were reverse
transcribed with biotin random primers, and serial ten-fold
dilutions of the cDNA, equivalent to 1 .mu.g, 100 ng and 10 ng of
starting RNA, were assessed by DECAL for differences in inhA and
ask/asd signals. The ten-fold differences in inhA mRNA and
four-fold differences in ask/asd mRNA could be easily detected even
in the highest cDNA dilution (FIG. 6). These results indicate that
DECAL is able to detect small differences in mRNA with limiting
amounts of RNA starting material. Furthermore, only 1% of the total
PCR probe generated from each tube pair was used in the experiment,
indicating that even lower limits of detection are likely.
[0070] C. Discussion
[0071] Current techniques to study differential gene expression in
bacteria are limited by the problems associated with separating
abundant rRNA sequences from mRNA, and by the difficulty of
achieving proportional amplification of sequences in complex PCR
reactions. The present study describes a simple and novel method
for studying differential gene regulation between two bacterial
populations. Differential gene expression is determined in a
straightforward manner by comparing the relative intensity with
which different PCR probes hybridize to individual colonies.
Simultaneous detection of multiple-genes can be performed,
identifying both mRNA sequences that are uniquely present in one
sample, and those that are present in both samples but unequally
represented. DECAL experiments are not dependent on polyA+ purified
mRNA that is lacking in prokaryotes, and can be performed without
customized arrays, and without knowledge of the entire bacterial
sequence. DECAL may also enhance the sensitivity of DNA array-based
detection methods by providing probes that can be PCR amplified
without significantly altering mRNA representation. DECAL should
extend the applicability of DNA arrays to investigations where
limited amounts of initial RNA is available.
[0072] Unlike total RNA or cDNA, customized amplification libraries
can be manipulated in a variety of ways to fulfill specific
requirements. For example, sets of CALs could be constructed that
contain only a subset of the entire genome. This could be easily
performed by using different restriction digests and more limited
size fractionation during CAL preparation. CALs with more limited
sequence representation might be advantageous when studying gene
expression in eukaryotic organisms with larger genomes. While CALs
require several weeks to construct, once prepared they are
available for many experiments. DECAL also has the unique ability
to allow unwanted RNA to be discarded without RNA subtraction
because only mRNA sequences that have complementary CAL sequences
can be represented in the final PCR probe. This property makes
DECAL ideally suited for in vivo investigations where RNA samples
may contain contaminating sequences from host tissue.
[0073] DECAL is critically dependent on removal of all
non-hybridizing CAL sequences. This problem was solved by the
development of a highly efficient wash protocol. During CAL
preparation, some genes flanking the ribosomal gene sequences are
removed along with the ribosomal coding cosmids, thus inevitably
some genes flanking the ribosomal gene sequences are removed along
with the ribosomal coding cosmids. However, cosmids with
overlapping inserts for CAL construction were used; therefore, only
a limited number of genes falling between the two Sau3A sites most
proximal to the ribosomal DNA sequences will have been removed
completely. Some genes may not have been digested into the 400 to
1,500 base pair fragments used in CAL construction, or may have
been lost during the pre-hybridization amplification step of CAL
synthesis. A more complete CAL could be constructed by combining
several digests made with different restriction enzymes.
[0074] DECAL was applied to study gene expression in M.
tuberculosis after treatment with the antibiotic isoniazid.
Isoniazid has long been a first line drug for the treatment of
tuberculosis (24) however, its full mechanism of action remains to
be established (25, 26). The discovery by the inventors of genes
that are induced by both isoniazid and ethambutol, two cell wall
active antibiotics that have different mechanisms of action (23,
25-28) adds further complexity to this issue. The role of the iniA
operon is not well understood. The phosphopantetheine attachment
site motif encoded by the iniA gene suggests that it encodes an
acyl carrier protein, however it may also have other functions.
Another acyl carrier protein acpM has been described recently that
both binds to and is induced by isoniazid (26). However no gene in
the iniA operon has significant homology to any gene in the operon
containing acpM or to the antigen 85 complex that has also been
shown to be induced by isoniazid (29). Unlike these genes, only
iniA is induced by both isoniazid and ethambutol. The inventors
speculate that the iniA operon may be induced as a protective
response to cell wall mediated cellular injury. If this is the
case, agents capable of blocking iniA, iniB, or iniC function would
be expected to act synergistically with isoniazid and other cell
wall active antibiotics to kill M. tuberculosis.
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