Lee Rowen

Complete sequence and gene map of a human major histocompatibility complex

Here we report the first complete sequence and gene map of a human major histocompatibility complex (MHC), a region on chromosome 6 which is essential to the immune system (reviewed in ref. 1). When it was discovered over 50 years ago the region was thought to specify histocompatibility genes, but their nature has been resolved only in the last two decades. Although many of the 224 identified gene loci (128 predicted to be expressed) are still of unknown function, we estimate that about 40% of the expressed genes have immune system function. Over 50% of the MHC has been sequenced twice, in different haplotypes, giving insight into the extraordinary polymorphism and evolution of this region. Several genes, particularly of the MHC class II and III regions, can be traced by sequence similarity and synteny to over 700 million years ago, clearly predating the emergence of the adaptive immune system some 400 million years ago. The sequence is expected to be invaluable for the identification of many common disease loci. In the past, the search for these loci has been hampered by the complexity of high gene density and linkage disequilibrium.Here we report the first complete sequence and gene map of a human major histocompatibility complex (MHC), a region on chromosome 6 which is essential to the immune system (reviewed in ref. 1). When it was discovered over 50 years ago the region was thought to specify histocompatibility genes, but their nature has been resolved only in the last two decades. Although many of the 224 identified gene loci (128 predicted to be expressed) are still of unknown function, we estimate that about 40% of the expressed genes have immune system function. Over 50% of the MHC has been sequenced twice, in different haplotypes, giving insight into the extraordinary polymorphism and evolution of this region. Several genes, particularly of the MHC class II and III regions, can be traced by sequence similarity and synteny to over 700 million years ago, dearly predating the emergence of the adaptive immune system some 400 million years ago. The sequence is expected to be invaluable for the identification of many common disease loci. In the past, the search for these loci has been hampered by the complexity of high gene density and linkage disequilibrium.Here we report the first complete sequence and gene map of a human major histocompatibility complex (MHC), a region on chromosome 6 which is essential to the immune system (reviewed in ref. 1). When it was discovered over 50 years ago the region was thought to specify histocompatibility genes, but their nature has been resolved only in the last two decades. Although many of the 224 identified gene loci (128 predicted to be expressed) are still of unknown function, we estimate that about 40% of the expressed genes have immune system function. Over 50% of the MHC has been sequenced twice, in different haplotypes, giving insight into the extraordinary polymorphism and evolution of this region. Several genes, particularly of the MHC class II and III regions, can be traced by sequence similarity and synteny to over 700 million years ago, clearly predating the emergence of the adaptive immune system some 400 million years ago. The sequence is expected to be invaluable for the identification of many common disease loci. In the past, the search for these loci has been hampered by the complexity of high gene density and linkage disequilibrium.

Analysis of Genetic Inheritance in a Family Quartet by Whole-Genome Sequencing

Runs in the Family The power to detect mutations involved in disease by genome sequencing is enhanced when combined with the ability to discover specific mutations that may have arisen between offspring and parents. Roach et al. (p. 636, published online 10 March) present the sequence of a family with two offspring affected with two genetic disorders: Miller syndrome and primary ciliary dyskinesia. Sequence analysis of the children and their parents not only showed that the intergenerational mutation rate was lower than anticipated but also revealed recombination sites and the occurrence of rare polymorphisms. Genomic sequencing of an entire family reveals the rate of spontaneous mutations in humans and identifies disease genes. We analyzed the whole-genome sequences of a family of four, consisting of two siblings and their parents. Family-based sequencing allowed us to delineate recombination sites precisely, identify 70% of the sequencing errors (resulting in > 99.999% accuracy), and identify very rare single-nucleotide polymorphisms. We also directly estimated a human intergeneration mutation rate of ~1.1 × 10−8 per position per haploid genome. Both offspring in this family have two recessive disorders: Miller syndrome, for which the gene was concurrently identified, and primary ciliary dyskinesia, for which causative genes have been previously identified. Family-based genome analysis enabled us to narrow the candidate genes for both of these Mendelian disorders to only four. Our results demonstrate the value of complete genome sequencing in families.

The complete 685-kilobase DNA sequence of the human β T cell receptor locus

The human β T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two types of coding elements—TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments) and eight trypsinogen genes—that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to be present in complementary DNA libraries.

Majority of divergence between closely related DNA samples is due to indels

It was recently shown that indels are responsible for more than twice as many unmatched nucleotides as are base substitutions between samples of chimpanzee and human DNA. A larger sample has now been examined and the result is similar. The number of indels is ≈1/12th of the number of base substitutions and the average length of the indels is 36 nt, including indels up to 10 kb. The ratio (Ru) of unpaired nucleotides attributable to indels to those attributable to substitutions is 3.0 for this 2 million-nt chimp DNA sample compared with human. There is similar evidence of a large value of Ru for sea urchins from the polymorphism of a sample of Strongylocentrotus purpuratus DNA (Ru = 3–4). Other work indicates that similarly, per nucleotide affected, large differences are seen for indels in the DNA polymorphism of the plant Arabidopsis thaliana (Ru = 51). For the insect Drosophila melanogaster a high value of Ru (4.5) has been determined. For the nematode Caenorhabditis elegans the polymorphism data are incomplete but high values of Ru are likely. Comparison of two strains of Escherichia coli O157:H7 shows a preponderance of indels. Because these six examples are from very distant systematic groups the implication is that in general, for alignments of closely related DNA, indels are responsible for many more unmatched nucleotides than are base substitutions. Human genetic evidence suggests that indels are a major source of gene defects, indicating that indels are a significant source of evolutionary change.

Comparative Genomics of the Human and Mouse T Cell Receptor Loci

The availability of the complete genomic sequences of the human and mouse T cell receptor loci opens up new opportunities for understanding T cell receptors (TCRs) and their genes. The full complement of TCR gene segments is finally known and should prove a valuable resource for supporting functional studies. A rational nomenclature system has been implemented and is widely available through IMGT and other public databases. Systematic comparisons of the genomic sequences within each locus, between loci, and across species enable precise analyses of the various diversification mechanisms and some regulatory signals. The genomic landscape of the TCR loci provides fundamental insights into TCR evolution as highly localized and tightly regulated gene families.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

BTL-II: A POLYMORPHIC LOCUS WITH HOMOLOGY TO THE BUTYROPHILIN GENE FAMILY, LOCATED AT THE BORDER OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II AND CLASS III REGIONS IN HUMAN AND MOUSE

Abstract Comparison of human and mouse genomic sequence at the border of the major histocompatibility complex (MHC) class II and class III regions revealed a locus encoding six exons with homology to the butyrophilin gene family and the location of a previously decribed gene, testis-specific basic protein (TSBP). We named the new locus BTL-II, for butyrophilin-like MHC class II associated. The six discernable exons of the BTL-II locus encode a small hydrophobic amino acid sequence (which may be a signal peptide), two immunoglobulin domains, a small 7-amino acid, heptad repeat-like exon, and a further two immunoglobulin domains. In mouse, an additional butyrophilin-like gene (NG10) is situated adjacent to BTL-II. Expression studies of the BTL-II locus in mouse showed that it is expressed in a range of gut tissues. We demonstrate that like many other genes from the MHC, BTL-II is polymorphic in a selection of diverse HLA haplotypes. In the light of the newly discovered locus, we revisit and discuss the possible origin of the butyrophilin gene family

Molecular Dissection of Prethymic Progenitor Entry into the T Lymphocyte Developmental Pathway

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-1 (TCF-1) (gene name Tcf7). To identify additional regulators of T cell specification, a cDNA library from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin−Sca-1+Kit+CD27−) and multipotent progenitors (Lin−Sca-1+Kit+CD27+), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bcl11b, TCF-1 (Tcf7), and HEBalt, Notch target Deltex1, Deltex3L, Fkbp5, Eva1, and Tmem131. Like GATA3 and Deltex1, Bcl11b, Fkbp5, and Eva1 were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only Bcl11b and HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative 1 and double negative 2) corresponding to T lineage specification. Bcl11b was uniquely T lineage restricted and induced by Notch/Delta signaling specifically upon entry into the T lineage differentiation pathway.

Analysis of the DNA sequence and duplication history of human chromosome 15

Here we present a finished sequence of human chromosome 15, together with a high-quality gene catalogue. As chromosome 15 is one of seven human chromosomes with a high rate of segmental duplication, we have carried out a detailed analysis of the duplication structure of the chromosome. Segmental duplications in chromosome 15 are largely clustered in two regions, on proximal and distal 15q; the proximal region is notable because recombination among the segmental duplications can result in deletions causing Prader-Willi and Angelman syndromes. Sequence analysis shows that the proximal and distal regions of 15q share extensive ancient similarity. Using a simple approach, we have been able to reconstruct many of the events by which the current duplication structure arose. We find that most of the intrachromosomal duplications seem to share a common ancestry. Finally, we demonstrate that some remaining gaps in the genome sequence are probably due to structural polymorphisms between haplotypes; this may explain a significant fraction of the gaps remaining in the human genome.

Differential Transcriptional Regulation of Individual TCR V Segments Before Gene Rearrangement

The promoter sequences of individual murine TCR Vβ segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCRβ 3′ enhancer. However, thymocytes of recombination-activating gene-2 (Rag2)-deficient mice allow the transcriptional activity of Vβ promoters to be studied before rearrangement. Here we report that many Vβ segments are detectably transcribed in Rag2−/− thymocytes and that there are significant differences in expression among different Vβ segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline Vβ transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal Vβ cluster (Vβ2.1, Vβ1.1, Vβ4.1) are relatively strongly expressed and more proximal Vβ segments (Vβ14.1, Vβ3.1, Vβ7.1, Vβ6.1) are only weakly expressed. Different Vβ segments also show different developmental programs of activation in different thymocyte subsets, with the Vβ5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag+ MHC class I−/− and class II−/− thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by β selection, before MHC-dependent selection. However, the expression pattern of Vβ2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.