Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen receptor beta chain genes.

The lineage of NK cells and their relationship to T lymphocytes have been controversial issues. Since rearrangement of the T cell antigen receptor beta chain genes occurs early in the ontogeny and differentiation of all T cells, this can be used as an unequivocal marker to discriminate T from non-T lymphocytes. Recent studies (16-18) examining T cell antigen receptor gene rearrangement and expression in certain IL-2-dependent NK cell lines and leukemias have revealed that some lines rearrange C beta genes, whereas others do not. However, it is important to establish whether these cell lines are representative of the major population of NK cells freshly derived from the host. Herein, we have purified granulocytes, CD16+ NK cells and T lymphocytes from human peripheral blood, prepared genomic DNA from each cell type, and then examined the organization of their T cell antigen receptor genes by restriction enzyme analysis using a C beta cDNA as probe. The C beta genes were in germline configuration in NK cells and granulocytes. In contrast, peripheral blood T lymphocytes showed rearrangement of the C beta gene. These data support the hypothesis that the majority of human peripheral blood NK cells are fundamentally distinct from T lymphocytes in lineage and nonself recognition.

The T cell antigen receptor complex expressed on normal peripheral blood CD4-, CD8- T lymphocytes. A CD3-associated disulfide-linked gamma chain heterodimer.

IL-2-dependent cell lines were established from normal peripheral blood T lymphocytes that express neither CD4 nor CD8 differentiation antigens. CD3+,4-,8- cell lines from 15 different donors failed to react with WT31, an mAb directed against the T cell antigen receptor alpha/beta heterodimer. Anti-Leu-4 mAb was used to isolate the CD3/T cell antigen receptor complex from 125I-labeled CD3+,4-,8- (WT31-) T cells. Using detergent conditions that preserved the CD3/T cell antigen receptor complex, an approximately 90 kD disulfide-linked heterodimer, composed of approximately 45- and approximately 40- (or approximately 37-) kD subunits, was coimmunoprecipitated with the invariant 20-29-kD CD3 complex. Analysis of these components by nonequilibrium pH gradient electrophoresis indicated that the approximately 40-kD and approximately 37-kD subunits were similar, and quite distinct from the more basic approximately 45-kD subunit. None of these three subunits reacted with an antibody directed against a beta chain framework epitope. Heteroantiserum against a T cell receptor gamma chain peptide specifically reacted with both the approximately 37- and approximately 40-kD CD3-associated proteins, but not with the approximately 45-kD subunit. CD3+,4-,8- cells failed to transcribe substantial amounts of functional 1.3-kb beta or 1.6-kb alpha mRNA, but produced abundant 1.6-kb gamma mRNA. Southern blot analysis revealed that these CD3+,4-,8- cell lines rearranged both gamma and beta genes, and indicated that the populations were polyclonal. The expression of a CD3-associated disulfide-linked heterodimer on CD3+,4-,8- T cell lines established from normal, adult peripheral blood contrasts with prior reports describing a CD3-associated non-disulfide-linked heterodimer on CD3+/WT31- cell lines established from thymus and peripheral blood obtained from patients with immunodeficiency diseases. We propose that this discrepancy may be explained by preferential usage of the two C gamma genes in T lymphocytes.

Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family.

Pathogenic mycobacteria, including the agent of tuberculosis, Mycobacterium tuberculosis, must replicate in macrophages for long-term persistence within their niche during chronic infection: organized collections of macrophages and lymphocytes called granulomas. We identified several genes preferentially expressed when Mycobacterium marinum, the cause of fish and amphibian tuberculosis, resides in host granulomas and/or macrophages. Two were homologs of M. tuberculosis PE/PE-PGRS genes, a family encoding numerous repetitive glycine-rich proteins of unknown function. Mutation of two PE-PGRS genes produced M. marinum strains incapable of replication in macrophages and with decreased persistence in granulomas. Our results establish a direct role in virulence for some PE-PGRS proteins.

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Characterization of a light-regulated gene encoding a new phycoerythrin-associated linker protein from the cyanobacterium Fremyella diplosiphon.

Cyanobacteria utilize multimeric protein complexes, the phycobilisomes, as their major light-harvesting antennae. Associated with the chromophorylated phycobiliproteins in these complexes are nonpigmented proteins, designated linker proteins. These linker proteins are believed to mediate assembly of the phycobilisome and energy transfer to the photosynthetic reaction center. We cloned and sequenced a gene, cpeE, encoding a previously uncharacterized linker protein which is expressed in green light in Fremyella diplosiphon. This gene is part of an operon containing two other phycoerythrin-associated linker genes, cpeC and cpeD. Transcription of the cpeCDE operon in green light results in two predominant species of mRNA of approximately 2,100 and 3,200 nucleotides. The shorter transcript encodes only CpeC and CpeD, while the longer contains the coding regions for all three linker proteins. By altering the pH of the resolving gel and the running buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this third linker protein CpeE can be resolved from the rod-core linker and the other rod linker proteins. The three proteins have an overall similarity of approximately 62%, and the genes encoding the three proteins are approximately 59% identical.

Characterization of the light-regulated operon encoding the phycoerythrin-associated linker proteins from the cyanobacterium Fremyella diplosiphon.

Many biological processes in photosynthetic organisms can be regulated by light quantity or light quality or both. A unique example of the effect of specific wavelengths of light on the composition of the photosynthetic apparatus occurs in cyanobacteria that undergo complementary chromatic adaptation. These organisms alter the composition of their light-harvesting organelle, the phycobilisome, and exhibit distinct morphological features as a function of the wavelength of incident light. Fremyella diplosiphon, a filamentous cyanobacterium, responds to green light by activating transcription of the cpeBA operon, which encodes the pigmented light-harvesting component phycoerythrin. We have isolated and determined the complete nucleotide sequence of another operon, cpeCD, that encodes the linker proteins associated with phycoerythrin hexamers in the phycobilisome. The cpeCD operon is activated in green light and expressed as two major transcripts with the same 5' start site but differing 3' ends. Analysis of the kinetics of transcript accumulation in cultures of F. diplosiphon shifted from red light to green light and vice versa shows that the cpeBA and cpeCD operons are regulated coordinately. A common 17-base-pair sequence is found upstream of the transcription start sites of both operons. A comparison of the predicted amino acid sequences of the phycoerythrin-associated linker proteins CpeC and CpeD with sequences of other previously characterized rod linker proteins shows 49 invariant residues, most of which are in the amino-terminal half of the proteins.

In vivo and in vitro footprinting of a light-regulated promoter in the cyanobacterium Fremyella diplosiphon.

Certain filamentous cyanobacteria, such as Fremyella diplosiphon, modulate the components of their light-harvesting complexes, the phycobilisomes, and undergo complex morphological changes in response to the wavelength of incident light, or light quality. The operon encoding the subunits of phycoerythrin, cpeBA, is transcriptionally activated in green light and is expressed at very low levels in red light. To begin elucidating the signal transduction pathway between the detection of specific light wavelengths and changes in gene expression, we have used in vivo footprinting to show that a protein is bound to the region upstream of the cpeBA transcription start site in both red and green light: two guanosine residues at -55 and -65 bp are protected from dimethyl sulfate modification in vivo. Using DNA mobility shift gel electrophoresis, we have shown that partially purified extracts of F. diplosiphon from both red and green light contain DNA-binding activity specific for the cpeBA promoter region. Using in vitro footprinting with dimethyl sulfate and DNase I, we have defined a binding site for this putative transcription factor, designated PepB (phycoerythrin promoter-binding protein), that extends from -67 to -45 bp on the upper strand and from -62 to -45 bp on the bottom strand, relative to the transcription start site. The binding site includes two hexameric direct repeats separated by 4 bp, TTGTTAN4TTGTTA. We conclude from these results that PepB is bound to the region upstream of the cpeBA promoter in F. diplosiphon in both red and green light. Therefore, additional factors or protein modifications must be required to allow light-regulated transcription of this operon.

DAtA: database of Arabidopsis thaliana annotation.

The Database of Arabidopsis thaliana Annotation (D At A) was created to enable easy access to and analysis of all the Arabidopsis genome project annotation. The database was constructed using the completed A.thaliana genomic sequence data currently in GenBank. An automated annotation process was used to predict coding sequences for GenBank records that do not include annotation. D At A also contains protein motifs and protein similarities derived from searches of the proteins in D At A with motif databases and the non-redundant protein database. The database is routinely updated to include new GenBank submissions for Arabidopsis genomic sequences and new Blast and protein motif search results. A web interface to D At A allows coding sequences to be searched by name, comment, blast similarity or motif field. In addition, browse options present lists of either all the protein names or identified motifs present in the sequenced A.thaliana genome. The database can be accessed at http://baggage. stanford.edu/group/arabprotein/

Changes in Accumulation and Synthesis of Transcripts Encoding Phycobilisome Components during Acclimation of Fremyella diplosiphon to Different Light Qualities.

We have used gene-specific DNA fragments as hybridization probes to quantitate the levels of transcripts encoding several phycobilisome polypeptides in the cyanobacterium Fremyella diplosiphon in response to changes in the light environment. While the levels of transcripts encoding allophycocyanin, the core linker polypeptide, and the constitutive phycocyanin subunits are similar in F. diplosiphon grown either in red or green light, the levels of other transcripts change dramatically. Transcripts encoding the inducible phycocyanin subunits are barely detected in green light-grown cells and very abundant in red light-grown cells, while the level of phycoerythrin mRNA is approximately 10-fold more in green than red light-grown cells. Quantitation of the phycoerythrin and inducible phycocyanin transcripts after transfer of cultures from green to red light and red to green light demonstrate that both increase rapidly upon exposure of cells to inductive illumination. The decrease in the phycoerythrin mRNA level in red light is much slower than the decline in the levels of the inducible phycocyanin transcripts in green light. Since the half-lives of the inducible phycocyanin and phycoerythrin transcripts do not change when F. diplosiphon is exposed to red or green illumination, the steady state levels of these mRNAs are primarily controlled by the rate of transcription. Therefore, the high level of phycoerythrin mRNA maintained for several hours after cultures are transferred from green to red illumination must result from continued transcription of the phycoerythrin gene set. Differences in expression from the phycoerythrin and inducible phycocyanin gene sets in response to light quality are discussed in terms of possible mechanisms involved in their regulation.

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex.

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination.

A gene essential for light-induced pigment production was isolated from the photochromogen Mycobacterium marinum by heterologous complementation of an M. marinum cosmid library in the nonchromogen Mycobacterium smegmatis. This gene is part of an operon and homologous to the Streptomyces griseus and Myxococcus xanthus crtB genes encoding phytoene synthase. Gene replacement at this locus was achieved via homologous recombination, demonstrating that its expression is essential for photochromogenicity. The ease of targeted gene disruption in this pathogenic Mycobacterium allows for the dissection of the molecular basis of mycobacterial pathogenesis.

Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification.

We have employed the technique of chromosome "walking" to determine the structure of 240 kilobases of amplified DNA surrounding the dihydrofolate reductase gene in methotrexate-resistant mouse cell lines. Within this region, we have found numerous DNA rearrangements which occurred during the amplification process. DNA subclones from regions flanking the dihydrofolate reductase gene were also utilized as hybridization probes in other cell lines. Our results show that: 1) amplification-specific DNA rearrangements or junctions are unique to each cell line; 2) within a given cell line, multiple amplification-specific DNA sequence rearrangements are found; 3) the degree of amplification of sequences flanking the dihydrofolate reductase gene shows quantitative variation among and within cell lines; and 4) both the arrangement of amplified sequences as well as the magnitude of gene amplification may vary with prolonged culture even under maintenance selection conditions. These studies indicate that there is no static repetitive unit amplified in these cells. Rather, a dynamic and complex arrangement of the amplified sequences exists which is continually changing.

An automated sample preparation system for large-scale DNA sequencing.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

High-resolution physical map of the Sinorhizobium meliloti 1021 pSyma megaplasmid.

To facilitate sequencing of the Sinorhizobium meliloti 1021 pSyma megaplasmid, a high-resolution map was constructed by ordering 113 overlapping bacterial artificial chromosome clones with 192 markers. The 157 anonymous sequence tagged site markers (81,072 bases) reveal hypothetical functions encoded by the replicon.

Infrequent genetic exchange and recombination in the mitochondrial genome of Candida albicans.

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.

The gamma T-cell antigen receptor.

The gamma-TCR is encoded by genes composed of V, J, and C elements that demonstrate a limited potential for recombinational diversity. These genes are rearranged, transcribed, and translated into proteins early during thymic ontogeny. Lymphocytes express gamma-TCR proteins on the plasma membrane only in association with the CD3 complex. gamma-TCR glycoproteins usually associate with another non-gamma glycoprotein, designated delta-TCR, to form a heterodimer receptor. Both non-disulfide-bonded and disulfide-bonded gamma/delta-TCR heterodimers have been identified on the plasma membrane of human T lymphocytes. On certain gamma-TCR-bearing T cell lines, a delta-TCR protein cannot be visualized by autoradiography. It is possible that delta-TCR proteins are associated with gamma-TCR glycoproteins on these cell lines but are not efficiently radiolabeled. Alternatively, it has been suggested that homodimers of gamma-TCR proteins can assemble with CD3 and be expressed on the plasma membrane of these cells. In adult lymphoid tissues, the majority of T lymphocytes expresses a CD3, alpha/beta antigen receptor, whereas only a minor subset (less than 5% of peripheral blood lymphocytes, lymph node, spleen, and thymocytes) express a CD3, gamma/delta antigen receptor. IL-2-dependent cell lines of both murine and human CD3, gamma/delta T cells have been established. Most CD3, gamma/delta T cell lines mediate cytotoxicity against a broad spectrum of tumor-cell targets, although the functional significance of this observation remains unclear. Cytotoxicity is apparently not restricted by or directed against MHC antigens. Antibodies against CD3 or gamma-TCR can induce proliferation and IL-2 secretion and can either augment or inhibit cytotoxicity, demonstrating that the gamma/delta-TCR is a functional receptor. The ligand recognized by this receptor has not been identified. The physiological role of T lymphocytes expressing gamma/delta-TCR, the molecular and structural properties of delta-TCR, and the relationship between CD3, alpha/beta T lymphocytes and CD3, gamma/delta T lymphocytes are the major unresolved questions that will be the primary focus of further experimentation.

Genomic evidence for a complete sexual cycle in Candida albicans.

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

Prevalence of small inversions in yeast gene order evolution.

Gene order evolution in two eukaryotes was studied by comparing the Saccharomyces cerevisiae genome sequence to extensive new data from whole-genome shotgun and cosmid sequencing of Candida albicans. Gene order is substantially different between these two yeasts, with only 9% of gene pairs that are adjacent in one species being conserved as adjacent in the other. Inversion of small segments of DNA, less than 10 genes long, has been a major cause of rearrangement, which means that even where a pair of genes has been conserved as adjacent, the transcriptional orientations of the two genes relative to one another are often different. We estimate that about 1,100 single-gene inversions have occurred since the divergence between these species. Other genes that are adjacent in one species are in the same neighborhood in the other, but their precise arrangement has been disrupted, probably by multiple successive multigene inversions. We estimate that gene adjacencies have been broken as frequently by local rearrangements as by chromosomal translocations or long-distance transpositions. A bias toward small inversions has been suggested by other studies on animals and plants and may be general among eukaryotes.