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Journal of Bacteriology, February 2001, p. 934-941, Vol. 183, No. 3
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.3.934-941.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742
Received 12 July 2000/Accepted 17 October 2000
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Neisserial lipooligosaccharide (LOS) contains three oligosaccharide
chains, termed the 
, 
, and 
chains. We used Southern hybridization experiments on DNA isolated from various
Neisseria spp. to determine if strains considered to be
nonpathogenic possessed DNA sequences homologous with genes involved in
the biosynthesis of these oligosaccharide chains. The presence or
absence of specific genes was compared to the LOS profiles expressed by
each strain, as characterized by their mobilities on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel and their reactivities
with various LOS-specific monoclonal antibodies. A great deal of
heterogeneity was seen with respect to the presence of genes encoding
glycosyltransferases in Neisseria. All pathogenic species
were found to possess DNA sequences homologous with the lgt
gene cluster, a group of genes needed for the synthesis of the chain. Some of these genes were also found to be present in strains
considered to be nonpathogenic, such as Neisseria lactamica, N. subflava, and N. sicca. Some nonpathogenic Neisseria spp. were able to express high-molecular-mass LOS
structures, even though they lacked the DNA sequences homologous with
rfaF, a gene whose product must act before gonococcal and
meningococcal LOS can be elongated. Using a PCR amplification strategy,
in combination with DNA sequencing, we demonstrated that N. subflava 44 possessed lgtA, lgtB, and
lgtE genes. The predicted amino acid sequence encoded by
each of these genes suggested that they encoded functional proteins;
however, structural analysis of LOS isolated from this strain indicated
that the bulk of its LOS was not modified by these gene products. This
suggests the existence of an additional regulatory mechanism that is
responsible for the limited expression of these genes in this strain.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Neisseria spp. are able to colonize a variety of human mucosal membranes, yet only a few species are considered to be pathogenic. The ability of Neisseria gonorrhoeae and N. meningitidis to cause disease depends on the expression of specific surface components, the absence of which correlates with a decreased or lack of ability to cause disease. In this context, commensal organisms probably fail to cause disease in healthy individuals because they lack one or more of the needed virulence determinants (6).
Lipooligosaccharide (LOS) is an important virulence determinant in N. gonorrhoeae and N. meningitidis. It is an outer membrane glycolipid involved in immune system evasion, attachment to epithelial tissue, host cell invasion, mediation of toxic damage in the fallopian tube, and stimulation of the production of bactericidal antibodies (2, 15, 20, 41, 44, 45). Biosynthesis of gonococcal and meningococcal LOS occurs via the same biosynthetic pathway producing a branched oligosaccharide attached to lipid A via two 3-deoxy-D-manno-2-octulosonic acid (KDO) molecules (12, 13, 21, 22, 48). The structure of LOS in the gonococcus and meningococcus shows a great deal of heterogeneity both within and among strains. Variation in the number of LOS components expressed and their relative concentrations and in the sugar compositions of the individual LOS molecules is observed (10, 12, 13, 21, 26, 27, 33, 47). The number of branches and the length of each oligosaccharide in the branch vary, and this variation is important in determining the pathogenic potential of the expressing strain (41, 47). Additionally, gonococci and meningococci can modify their LOS by adding a sialic acid residue onto a terminal galactose that is found in the lacto-N-neotetraose structure (32). This addition effectively masks the LOS molecule from host immune functions, making the organism resistant to complement-mediated killing, i.e., serum resistant (29). Certain LOS structural features can be deduced from their ability to react with monoclonal antibodies (MAbs) (9, 27, 28) and their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (19, 43).
In recent years, several LOS biosynthesis genes have been cloned and
characterized. The rfaC gene product adds the first heptose (Hep I) to KDO (50). The rfaF gene product adds
the second Hep onto Hep I (35, 40) and is required for
-chain elongation (12, 34). The first Glc of the chain is added by gene lgtF (24). Genes
lgtA, lgtB, lgtC, lgtD, and lgtE (called
lgt locus or lgt gene cluster) are responsible
for the synthesis of different chains (14). Genes
lgtA, lgtC, and lgtD contain poly(G) tracts (5, 7, 49). When the number of guanines found in these genes changes during DNA replication, alterations in the coding sequence may occur, making translation of the proteins encoded by these
genes susceptible to premature termination. Loss of function of any of
these genes effects changes in the structure of LOS. Similarly,
lgtG, required for addition of the first Glc of the chain, contains a poly(C) tract. When this gene encodes a functional protein, strains can extend the chain (3). The chain may be composed of a single glucose or lactose or lactose with
additional sugars (13, 47, 48). Finally, the
rfaK gene product is required for -chain elongation
(23, 24).
While commensal Neisseria spp. are rarely associated with disease, some strains occasionally cause clinical illnesses (e.g., endocarditis caused by N. sicca (11) and arthritis caused by N. subflava (1). We have recently shown that the expression of the lacto-N-neotetraose LOS is able to promote gonococcal invasion into tissue culture cells (41), indicating that the expression of the lacto-N-neotetraose glycoform is important for the establishment of invasive infections. Since most commensal Neisseria spp. express LOS molecules that fail to bind LOS-specific MAbs that recognize this LOS structure, we hypothesized that commensal strains lack the genes needed to make this LOS moiety. Since LOS plays such a prominent role in gonococcal and meningococcal disease, we believe that commensal Neisseria spp. rarely cause disease because they lack the ability to make this essential virulence component. This study was designed to test the hypothesis that LOS-biosynthetic pathways utilized by pathogenic strains are conserved within the Neisseriaceae. In this study, we used several cloned gonococcal LOS-biosynthetic genes as probes to determine if nonpathogenic Neisseria spp. possess homologous genes. Because N. subflava 44 was found to possess genes with homology to several LOS-biosynthetic genes in this study yet failed to produce the expected LOS molecules (42), we studied the organization and expression of some of its LOS-biosynthetic genes in greater detail.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Bacterial strains, plasmids, oligonucleotides, and culture
conditions.
All bacterial strains, plasmids, and oligonucleotide
primers used in this study are listed in Tables
1 and 2.
All Neisseria strains were grown in phosphate-buffered
gonococcal broth plus growth supplements (46)-0.042%
sodium bicarbonate or on gonococcal agar base (Difco) plus growth
supplements (46) in a 37°C CO2 incubator.
Escherichia coli strains were grown on Luria-Bertani plates
(39). Kanamycin was used at 30 µg/ml, and X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) was
used at 35 µg/ml.
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Chemicals, reagents, and enzymes. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, Mass.). All chemicals used for this study were reagent grade or better and were purchased from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise specified. MAbs 2-1-L8, 25-1-LC1, and 17-1-L1 were generously provided by Wendell Zollinger, Walter Reed Army Institute of Research, Washington, D.C. MAb 1B2 was a gift from J. McLeod Griffiss, University of California, San Francisco. MAb 3G9 was graciously provided by Peter Rice, Boston University, Boston, Mass. MAb B5 was a gift from Margaret A. Gidney, Institute for Biological Sciences, National Research Council, Ottawa, Canada.
DNA isolation procedures. Chromosomal DNA was isolated by a modification of the method of Rodriguez and Tait (38). This modification involved the use of proteinase K (Fc, 50 pg/ml) instead of pronase and subsequent incubation at 37°C overnight. Plasmid DNA was isolated by the alkaline lysis procedure of Birnboim and Doly (4).
Southern hybridization experiments. Southern analysis was performed as described by Sambrook et al. (39) using the Genius 7 nonradioactive nucleic acid labeling kit of Boehringer Mannheim (Indianapolis, Ind.). The probe comprising the sequence from the 3' end of rfaF was made from a 577-bp StuI-EcoRI fragment from pRDM45 (8). The probes for lgtF and rfaK were made by PCR amplification of N. gonorrhoeae F62 chromosome DNA with primers rfak-147F and rfak-3780R. The PCR product was cloned into pK18up (pK18 [37] modified by the addition of a DNA sequence [using primers uptake-A and uptake-B, each containing a gonococcal transformation uptake sequence and an EcoRI site] to a pK18 BglII site), giving pRFAK2-1. This plasmid was digested with EcoRI, generating three fragments: 0.8 kb of lgtF, 1.6 kb of rfaK, and 2.1 kb of the vector. The fragments were separated on a 1.2% agarose gel by electrophoresis in Tris-acetate buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA [pH 8.1]), and the appropriate fragment was purified from the gel and labeled for Southern hybridization. Other probes were made by labeling PCR products directly.
SDS-PAGE analysis. Proteinase K-treated whole-cell lysates were prepared from 18- to 20-h cultures by the procedure of Hitchcock and Brown (19). Lysates containing approximately 200 ng of LOS were subjected to SDS-PAGE either on a 13% acrylamide gel in a Tris-glycine buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3) at a constant current of 30 mA per gel for ~4 h at 10°C or on a Tris-Tricine gel (16.5%; Bio-Rad) in Tris-Tricine buffer in accordance with the protocol suggested by the manufacturer. Gels were fixed overnight in 40% ethanol-5% acetic acid, and the LOS was visualized by silver staining (43).
PCR. PCR was used to generate the DNA fragments employed in the Southern hybridization and gene cloning experiments. DNA amplifications were performed using the GeneAmp PCR kit from Perkin-Elmer Cetus (Norwalk, Conn.). PCR to amplify the lgtA-E gene cluster was performed using the Expand PCR kit from Boehringer Mannheim under recommended conditions. Primers were obtained from either the University of Maryland Protein and Nucleic Acids Facility or Bioserve, Inc. (Laurel, Md.).
Western blot and colony blot analyses. After SDS-PAGE, LOSs were electrotransferred onto an Immobilon-P membrane (Millipore Corp., Bedford, Mass.) in a Tris-glycine-methanol buffer (0.025 M Tris, 0.192 M glycine, 20% methanol) at a constant voltage of 100 V for 1 h in accordance with the protocol provided by Bio-Rad Corp. After being air dried for 1 h, the membrane was processed by the same procedure as that used for colony blotting, which was described previously (40).
Transformation.
Recombinant DNA transformation into E. coli DH5MCR was done according to the standard protocols
(39). Recombinant DNA transformation into N. gonorrhoeae F62 was done by spot transformation (18). After overnight growth, colonies were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, N.H.) and screened for reactivity to MAb 2-1-L8.
Recombinant DNA methods.
Primers JL50 and Got5220R were used
to amplify N. subflava 44 chromosome DNA via PCR. The 3-kb
PCR product was digested with EcoRI and BamHI and
inserted into the corresponding sites in pK18. The recombinant plasmid
was named as pNS44lgtABE. Another plasmid, pNS44lgtAB
E, was generated by amplifying a
fragment of N. subflava 44 chromosome DNA by PCR using
primers JL50 and DA5, digesting the amplicon with EcoRI and
AgeI, and cloning the fragment into pK18. Primers
196-N-1040R and lgtG-1260R were used to amplify the lgtG
gene of N. subflava 44. The 1.2-kb fragment was cloned into
pK18 and named pNS44lgtG. All of these recombinant plasmids (except pNS44lgtABE) were sequenced by the DNA
Sequence Facility/Center for Agricultural Biotechnology, University of
Maryland, College Park.
Nucleotide sequence accession numbers. The DNA sequences of lgtABE and lgtG from N. subflava 44 appear in GenBank under accession no. AF240672 and AF241526, respectively.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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SDS-PAGE profiles of various Neisseria strains.
Neisserial LOS exhibits significant heterogeneity when analyzed by
SDS-PAGE. The data presented in Fig. 1
show the SDS-PAGE profiles of LOSs expressed by several commensal
species. These data demonstrate that all of the strains examined
express LOS molecules that are larger than the LOS made by FA5100 (Fig.
1, lane 10). Since the structure of LOS made by FA5100 contains a single Hep added onto the lipid A-KDO core (12), we
concluded that the LOSs isolated from all of the strains we examined
must possess additional sugars.
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chain
[16]), MAb 3G9 (marker for a lactosyl group on the chain [17]), MAb 17-1-L1 (marker for an alternate
-chain extension terminating in galactose [16]), MAb
25-1-LC1 (marker for the presence of -chain glucose and -chain
lactose or sucrose [42]), and MAb B5 (marker for the
presence of phosphoethanolamine on Hep II [36]). Of the
nonpathogenic strains examined, N. lactamica 5841 contained LOS components that bound MAbs 1B2 and 2-1-L8 (Table
3). N. subflava 44 bound MAb
1B2 weakly on the colony blot, but we were able to visualize binding to
a specific LOS band only when the SDS-PAGE gel was grossly overloaded
(data not shown). These data suggest that LOSs isolated from most
commensal organisms are significantly different structurally from LOSs
isolated from pathogenic strains.
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Determination of the presence of rfaF.
One of the
key steps in the externalization of gonococcal LOS is the addition of
Hep II onto Hep I: failure to make a diheptosyl structure results in
the expression of LOS molecules that are terminated after a single Hep.
In order for the gonococcus to express an LOS molecule that has sugar
additions onto C-4 of Hep I, it must express rfaF (34,
40). If all Neisseria spp. express their LOSs using
the same biosynthetic pathway, then homologs to rfaF should
be found in all strains. Southern hybridizations were conducted on
HincII-digested chromosomal DNA under conditions that should
detect DNA sequences with limited homology to rfaF. The data
presented in Fig. 2 indicate that DNA
sequences with significant homology to the probe were found in about
half of the strains examined. While all of the gonococcal strains,
N. meningitidis 891, N. lactamica 5841, N. subflava 44, N. sicca 342, and the N. cinerea strains, possessed this gene, N. subflava 52, 4324, 4325, and 4327, N. sicca 4318, and the N. flavescens strains lacked this gene. If the rfaF gene
in N. subflava 52, 4324, 4325, and 4327, N. sicca
4318, and the N. flavescens strains were to encode a
functional protein and if the appropriate biosynthetic precursor were
made, these strains should produce LOS that contains at least a
diheptosyl structure linked to KDO. These strains should then make an
LOS with an apparent mobility greater than that seen for strain FA5100.
The data presented in Fig. 1 and 2 demonstrate that all strains that
contained the DNA sequence that corresponded to rfaF
expressed LOSs with higher molecular weights. Since all of the strains
that lacked DNA sequences homologous to rfaF were also able
to make LOSs that were significantly larger than the LOS made by strain
FA5100, a strain whose LOS possesses a single Hep (12),
this indicates that sugars can be added onto Hep I in the absence of
the rfaF gene product or that a nonhomologous functional
equivalent to rfaF exists in these strains.
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Determination of the presence of the lgt gene
cluster.
Most of the genes required for the synthesis of the
-chain oligosaccharide in N. gonorrhoeae are found in a
gene cluster consisting of lgtA-E (7, 14). We
used a probe containing DNA sequences homologous to lgtA and
D to test for their presence in the various
Neisseria spp. We hypothesized that strains that possess
these sequences would have the genetic potential to express the
lacto-N-neotetraose and related structures and, as such,
could make LOS components that react with the appropriate MAbs. A
954-bp fragment internal to lgtA was used to screen for the
presence of lgtA and/or lgtD by Southern
hybridization. The probe used contained all of lgtA except
the first 49 bp and the last 45 bp. Under the stringency conditions
employed, both lgtA and lgtD would bind the
probe, due to the high degree of homology between the two genes.
However, the binding to lgtA would be more efficient than
the binding to lgtD due to the greater sequence identity between lgtA and the probe. Strains that failed to bind the
probe were presumed to lack both lgtA and lgtD.
Correlation of genetic data with SDS-PAGE profiles and MAb binding. The presence of LOS components able to bind LOS-specific MAbs correlated with the presence or absence of genes located within the lgt gene cluster for all strains examined, with the exception of strains N. subflava 44 and N. sicca 342 (Table 3). While we were able to show that N. subflava 44 and N. sicca 342 contained DNA sequences with high homology to the lgt gene cluster, their LOS SDS-PAGE profiles (Fig. 1) and their lack of ability to bind LOS-specific MAbs suggested that the genes were nonfunctional.
Analysis of N. subflava 44 for the presence of other
biosynthetic genes.
We chose to examine the genetic potential of
N. subflava 44 to make LOS in greater detail because its LOS
was able to bind MAb 1B2 on a colony blot yet failed to express a
detectable amount of the lacto-N-neotetraose LOS component
when its LOS was analyzed by SDS-PAGE. Furthermore, mass spectrometry
combined with fluorophore-assisted carbohydrate electrophoresis
monosaccharide composition analysis showed that N. subflava
44 expresses two major LOS components (LOSI and LOSII)
(42). LOSI contains one glucose on both the and chains. LOSII is structurally related to LOSI and differs from it by
the addition of a hexose (either glucose or galactose) on the chain. Only a trace amount of LOS expressed in N. subflava 44 could further extend the chain to form the
lacto-N-neotetraose structure that could be detected on a
colony blot. Based on our structural analysis of N. subflava
44 LOS (42), the structure of its core is identical to the
gonococcal and meningococcal LOS core structures. In the gonococcus and
the meningococcus, the proteins encoded by lgtF and
rfaK are necessary for making the core LOS structure
(24). Additional Southern hybridization experiments were
performed on chromosomal DNA isolated from N. subflava 44. The data indicated that it contained DNA sequences that bound lgtF- and rfaK-specific probes with the same
intensity as the other tested gonococcal strains, indicating that this
strain possessed these genes (data not shown).
Analysis of the lgt gene cluster from N. subflava 44. To study why N. subflava 44 only expressed a limited amount of lacto-N-neotetraose LOS, we characterized the lgt gene cluster. We used the DNA sequence of the N. gonorrhoeae F62 lgt gene cluster to design a pair of primers capable of amplifying this region from strain N. subflava 44 and cloned the amplicon into plasmid pK18 (named pNS44lgtABE). DNA sequence analysis of the PCR-amplified DNA showed that it contained three genes, corresponding to lgtA, lgtB, and lgtE. Each of these genes was >90% identical at the DNA level to its homologs found in N. gonorrhoeae and N. meningitidis. The predicted amino acid sequence of each of the three proteins possessed significant amino acid sequence identity to their homologs in N. meningitidis MC58, N. meningitidis 126E, and N. gonorrhoeae F62 (>98% identical) (data not shown).
LgtE is responsible for the addition of galactose onto the chain
(49). In order to determine if
lgtENS (the lgtE gene isolated from
N. subflava 44) was functional, we used the spot transformation procedure to introduce lgtENS
(contained on pNS44lgtABE) into N. gonorrhoeae
F62 lgtA lgtE. We identified a transformant that acquired reactivity with MAb 2-1-L8 (named F62TF#1). The region
surrounding these loci was amplified using PCR, and the DNA sequence of
the amplicon was determined. The genetic organization of the
recombinant, shown in Fig. 3, indicates
that we not only replaced lgtEF62 (the
lgtE gene isolated from N. gonorrhoeae F62) with
lgtENS but also replaced
lgtBF62 with lgtBNS. The
SDS-PAGE profile of LOS expressed by the transformant (Fig.
4, lanes 4 and 5) indicates that, when
lgtENS is introduced into N. gonorrhoeae F62 lgtA lgtE,
LgtENS is functional.
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lgtE) was used to transform
N. gonorrhoeae F62 lgtA by the spot
transformation procedure. Many transformants that had lost the ability
to bind MAb 2-1-L8 and that had acquired reactivity with MAb 1B2 were identified, indicating that lgtANS encodes a
functional protein. The complete replacement of
lgtAF62 with lgtANS was
verified by PCR amplification and DNA sequence analysis. Functional
complementation of lgtAF62 with
lgtANS was verified by examination of the LOS phenotype by SDS-PAGE (Fig. 4, lanes 2 and 3). These data indicate that
the minor sequence differences between lgtANS
and lgtAGC (the lgtA gene isolated
from gonococci) do not have an effect on LgtA function. They also
suggest that lgtANS should encode a functional
LgtA protein in N. sublflava 44.
Determination of the presence of lgtG in N. subflava 44.
Structural analysis showed that the major LOSs
expressed by N. sublflava 44 possess one glucose on the chain (42). Banerjee et al. (3) recently
described gene lgtG, whose product adds a glucose onto Hep
II. We used a probe specific for this gene in Southern hybridization
experiments. These experiments indicated that N. subflava 44 possessed DNA sequences homologous to lgtG (Table 3). The
region corresponding to lgtG was amplified using PCR, the
amplicon was cloned, and the sequence of the amplified DNA was
determined. DNA sequence analysis indicated that this gene contained a
polycytosine tract that would result in the expression of a functional
protein. Comparison of the amino acid sequence of LgtG from strain
15253 to that of LgtG from N. subflava 44 indicated that
these two proteins were identical, with the exception of some minor
sequence differences in the first 20 amino acids. Additionally, from
the predicted coding sequences, lgtGNS initiates expression from a TTG start codon, while
lgtG15253 initiates expression from an ATG.
Since structural studies have demonstrated the presence of a glucose in
N. subflava 44 LOS (42), this indicates that lgtGNS is functional.
Correlation of Southern hybridization data with SDS-PAGE
analysis.
Kim et al. (25) showed that N. lactamica LOS was immunologically related to gonococcal and
meningococcal LOS. The data presented in Table 3 indicate that N. lactamica 5841 possesses DNA sequences homologous to the
lgt gene cluster. This provides genetic evidence to support
the immunological observations of Kim et al. (25). The
failure of the two N. cinerea strains that we examined to bind the LOS-specific MAbs used as markers for -chain extensions was
expected since these strains lacked the DNA sequences homologous to the
lgt gene cluster. Kim et al. (25) were able to
identify strains of N. cinerea that reacted with MAb 3F11
(this antibody has the same specificity as MAb 1B2 used in this study).
The ability of N. cinerea 32824 to bind MAb 25-1-LC1
suggests that this strain has a functional lgtG gene. The
observations of Kim et al. (25), combined with our data,
provide one genetic explanation for the structural variation in LOS
seen in this species (i.e., the deletion or acquisition of the needed
coding region).
-chain structures (30). Likewise, several
gonococcal strains that express additional LOS structures have been
identified (13).
Speciation of the Neisseriaceae has been based on phenotypic
characteristics, including the ability to produce pigment, patterns of
acid production from carbohydrates, production of a polysaccharide from
sucrose, reduction of nitrate to nitrite, and the production of
deoxyribonucleases (31). However, since many examples of localized interspecies recombination between the pathogenic and commensal species have been shown to have occurred (51)
and since some commensal organisms are occasionally associated with disease, it seems possible that these apparent discrepancies in the
presence of LOS-biosynthetic genes seen for some species can be
attributed to the inherent genetic instability of the genus. The
ability of some strains of N. subflava and N. sicca to express high-molecular-mass LOS in the absence of an
rfaF homolog suggests that some of these species have
acquired the ability to express alternate LOS structures.
Taking into account all the data, we have concluded that our current
view of the biosynthetic potential and types of LOS structures that
might be made by the Neisseriaceae is too limited. This has significant ramifications for researchers as they try to elucidate the
role of LOS in the disease process, because they may inadvertently choose the wrong strain for their in vitro models and thereby conclude
that a particular molecule is not important in the disease process,
because their strain lacked the genetic capability to make the
important variation of that structure. The nonpathogenic Neisseriaceae may act as a reservoir for LOS antigenic
variants and thereby indirectly contribute to disease.
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ACKNOWLEDGMENTS |
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We thank the members of the laboratory, especially James Levin, for all of their help.
This work was supported by a grant from the National Institutes of Health to D.C.S. (grant A124452). Dan Arking was supported by a grant from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742. Phone: (301) 405-5448. Fax: (301) 314-9489. E-mail: DS64{at}UMAIL.UMD.EDU.
Present address: Institute for Genetic Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD 21205.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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