Divergent evolution of T cell repertoires: extensive diversity and developmentally regulated expression of the sheep gamma delta T cell receptor.

Sheep gamma delta T cells express an unprecedented repertoire of antigen receptors contributed by increased diversity in both variable and constant region gene segments. Variable region diversity results mainly from the utilization of a large family of duplicated V delta genes that have retained two distinct hypervariable segments comparable with the complementarity determining regions present in other antigen receptor V genes. This implies that sheep V delta chains have been intensely selected during evolution, probably at sites involved in ligand recognition. The sheep gamma delta heterodimer occurs in at least five isotypic variants formed by the association of a single C delta segment with one of five functional C gamma segments, each with distinctive hinge regions. Our analysis also shows that the establishment of a normal peripheral repertoire is both developmentally regulated and dependent on the continual presence of a functional thymus during ontogeny. The existence of an expanded V gene repertoire and multiple receptor isotypes together with the prominence of gamma delta T cells in the sheep immune system argues that this lineage of T cells has a more elaborate functional role in this evolutionary pathway.     

Mosaic evolution of ruminant stomach lysozyme genes

The genomes of ruminant artiodactyls, such as cow and sheep, have approximately 10 lysozyme genes, 4 of which are expressed in the stomach. Most of the duplications of the lysozyme genes occurred 40-50 million years ago, before the divergence of cow and sheep. Despite this, the coding regions of stomach lysozyme genes within a species (e.g., cow, sheep, or deer) are more similar to each other than to lysozyme genes in other ruminants. This observation suggests that the coding regions of the stomach lysozyme genes have evolved in a concerted fashion. Our previous characterization of 3 cow stomach lysozyme genes suggested that it was only the coding exons that had participated in concerted evolution. To determine whether the introns and flanking regions of ruminant stomach lysozyme genes are evolving in a concerted or a divergent fashion, we have isolated and characterized 2 sheep stomach lysozyme genes. Comparison of the sequences of the sheep and cow stomach lysozyme genes clearly shows that the introns and flanking regions have evolved, like the 3' untranslated region of the mRNAs, in a divergent manner. Thus, if the four coding exons are evolving by concerted evolution, then a mosaic pattern of concerted and divergent evolution is occurring in these genes. The independent concerted evolution of coding exons of the ruminant stomach lysozyme gene may have assisted in the accelerated adaptive evolution of the lysozyme to new function in the early ruminant.     

The repair and recombination enzyme ERCC1 is not required for immunoglobulin class switching

Class switch recombination (CSR) is a programmed gene rearrangement in which a B cell which is producing IgM and IgD antibody develops into an IgG-, IgA- or IgE-expressing cell. This is achieved by recombination between switch regions located 5' of each of the immunoglobulin heavy chain constant regions, except Cdelta. The mechanism of CSR has not been resolved but it is thought to involve a double-strand break followed by end joining. It has previously been suggested that the nucleotide excision repair protein ERCC1 may be involved in CSR due to its known roles in removal of 3' single-stranded tails in various types of recombination. In this study, we examined class switching in cultured splenocytes from ERCC1-deficient mice and found no evidence of any deficiency.     

Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.     

Immunoglobulin D switching can occur through homologous recombination in human B cells.

Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.     

The nucleotide sequence of a human immunoglobulin C gamma1 gene

We report the nucleotide sequence of a gene encoding the constant region of a human immunoglobulin gamma 1 heavy chain (C gamma 1). A comparison of this sequence with those of the C gamma 2 and C gamma 4 genes reveals that these three human C gamma genes share considerable homology in both coding and noncoding regions. The nucleotide sequence differences indicate that these genes diverged from one another approximately 608 million years ago. An examination of hinge exons shows that these coding regions have evolved more rapidly than any other areas of the C gamma genes in terms of both base substitution and deletion/insertion events. Coding sequence diversity also is observed in areas of CH domains which border the hinge.     

The ubiquitously expressed DNA-binding protein late SV40 factor binds Ig switch regions and represses class switching to IgA

Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery.     

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.     

Products and implied mechanism of H chain switch recombination

The Ig H chain switch is a DNA recombination event. The recombination occurs between two or more switch regions, areas of tandem sequence duplication that lie upstream of the corresponding H chain C region genes. We have determined the DNA sequence at four recombination sites in three molecularly cloned, rearranged switch regions. All eight donor and recipient recombination sites are at the common pentamers GGGGT, GAGCT, and GGTGG. One of the switch recombination events is an inversion of S gamma 3 sequences. Another of the recombinational events is an internal S gamma 1 deletion, which may be switch enzyme mediated. These results, together with other switch recombination site sequences, suggest that switch recombination is mediated by cutting enzymes with modest specificity and religation enzymes with no specificity.     

A comparative genomic analysis of the cow,pig, and human CFTR genes identifies potential intronic regulatory elements

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.     

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.     

Investigating ancient duplication events in the Arabidopsis genome

The complete genomic analysis of Arabidopsis thaliana has shown that a major fraction of the genome consists of paralogous genes that probably originated through one or more ancient large-scale gene or genome duplication events. However, the number and timing of these duplications still remains unclear, and several different hypotheses have been put forward recently. Here, we reanalyzed duplicated blocks found in the Arabidopsis genome described previously and determined their date of divergence based on silent substitution estimations between the paralogous genes and, where possible, by phylogenetic reconstruction. We show that methods based on averaging protein distances of heterogeneous classes of duplicated genes lead to unreliable conclusions and that a large fraction of blocks duplicated much more recently than assumed previously. We found clear evidence for one large-scale gene or even complete genome duplication event somewhere between 70 to 90 million years ago. Traces pointing to a much older (probably more than 200 million years) large-scale gene duplication event could be detected. However, for now it is impossible to conclude whether these old duplicates are the result of one or more large-scale gene duplication events. abbreviations dA, fraction of amino acid substitutions; Kn, number of nonsynonymous substitutions per nonsynonymous site; Ks, number of synonymous substitutions per synonymous site; MYA, million years ago     

Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals

Approximately 15 different alpha, or CXC, chemokines have thus far been isolated from 11 species of mammals. Among the best studied chemokines are the 12 human proteins that are encoded by 11 paralogous genes. In order to better understand the evolution and function of this group of genes, we isolated and characterized six novel GRO and GRO-related cDNA sequences from the cow (Bos taurus), the sheep (Ovis aries), the rabbit (Oryctolagus cuniculus), and the guinea pig (Cavia porcellus). The amino acid sequence of the diverged guinea pig GRO or KC gene is only 50%-60% similar to presumed orthologs from other species, while the sheep and cow GRO proteins are 90%-99% similar to each other. The presence of multiple GRO genes in the cow, the rabbit, and the sheep is consistent with what has been observed for humans. Phylogenetic analyses of amino acid sequences from 44 proteins indicate that genes orthologous to many of the 11 known from humans exist in other species. One such gene, interleukin 8, or IL8, has been isolated from nine species, including the rodent guinea pig; however, this gene is absent in the rat and the mouse, indicating a unique gene loss event in the rat/mouse (muroid rodent) lineage. The KC (or MIP2) gene of rodents appears to be orthologous to the GRO gene found in other taxonomic orders. Combined evidence from different sources suggests that IP10 and MIG share sister taxon relationships on the evolutionary tree, while the remaining paralogous genes represent independent lineages, with limited evidence for kinship between them. This observation indicates that these genes originated nearly contemporaneously via a series of gene duplication events. Relative-rate tests for synonymous and nonsynonymous nucleotide substitutions in the KC and IL8 genes did not detect rate heterogeneity; however, there are several notable features regarding the IL8 genes. For example, the IL8 proteins from two Old World monkeys are as similar to one another as they are to the IL8 protein from humans, and all observed nucleotide differences between the IL8 genes of the two monkeys cause amino acid changes; in other words, there are no synonymous differences between them.