The newly isolated region encompasses the 170-kb DNA upstream of the V3-64 segment and its J H-distal end hybridized with a human telomere repeat probe. In this study, we further extended the region by screening and characterization of human YAC, P1, and cosmid clones (Fig. 64 V H segments (V6-1 to V3-64) have been completely sequenced and were categorized into 33 structurally functional and 31 pseudo V H segments ( 8). Previously, we isolated and analyzed the J H-distal 0.8-Mb region of the human V H locus. Identification of Nonimmunoglobulin and Repetitive Sequences. Accuracy of the nucleotide sequence was estimated to be 99.98% by comparison of the two sequences of 23-kb DNA between the V6-1 segment and D gene cluster from two independent cosmid clones. ( c) The remaining gaps between contigs were then filled by primer walking using the plasmid DNA of the shotgun clones that bridge different contigs as a template.
Nucleotide sequence software#
The 192 sequences obtained were assembled to generate contigs by Sequencher software (Gene Codes Corporation, Inc., Ann Arbor, MI). The remaining DNAs were sequenced by a combination of shotgun and primer walking methods as follows ( a) insert DNA (average size ∼3–4 kb) for the shotgun libraries was obtained from cosmid and P1 clones either by partial digestion with Sau3AI or by mechanical shirring and subsequent fractionation by agarose gel electrophoresis ( b) plasmid DNA of 96 shotgun clones from each cosmid or P1 clone was used for the first round sequencing analysis by using vector primers of both ends. 637-kb regions whose plasmid subclones were available were sequenced by a primer walking method. Two different methods were used to determine the nucleotide sequence. Although these molecules are likely to be derived from a common ancestral receptor molecule, much more complex molecular mechanisms are used for the refinement of the V-region repertoire of Ig than TCR after maturation of lymphocytes. In contrast, the V-region diversity of TCR is fixed through the selection process in the thymus and maintained without any modification ( 3). Upon encounter with antigens, further diversification and refinement of the Ig repertoire is accomplished by a process known as affinity maturation, which includes somatic hypermutation, receptor editing, somatic gene conversion, and clonal selection ( 1, 2). Second, during the ontogeny of lymphocytes, each one of these segments is chosen to undergo a somatic recombination event called V-(D)-J recombination, giving rise to the combinatorial and junctional amino acid diversity. First, the V regions are encoded by two or three genetic segments, namely V, D, and J segments, each of which comprises multiple copies and provides the repertoire before somatic mutation. Generation of the primary V-region repertoire depends on the common genetic basis and molecular mechanisms characteristic of these antigen-receptor molecules ( 1– 3). The NH 2-terminal portion of their subunits is called the V region because of its diverse amino acid sequence required for interaction with a diverse spectrum of antigens. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.ĭuring the vertebrate immune response, Ig and TCR play central roles in antigen recognition. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. However, an independent branch in the tree contained a single V H, V4-44.1P, sharing similar levels of homology to human V H families and to those of other vertebrates. Phylogenetic analysis of 114 V H segments clearly showed clustering of the V H segments of each family. Conservation of the promoter and recombination signal sequences was observed to be higher in functional V H segments than in pseudogenes. Combinatorial diversity of V H region was calculated to be ∼6,000. Of the 44 V H segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. The region contains 123 V H segments classifiable into seven different families, of which 79 are pseudogenes. The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (V H) region locus was determined and 43 novel V H segments were identified.
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