Nucleotide sequence of a cDNA encoding the constant region of a rabbit immunoglobulin light chain of the lambda type

A cDNA library has been constructed in the plasmid pBR322 using as template 12 S poly(A)-RNA isolated from spleen cells of a hyperimmunized Basilea rabbit. One of these cDNA-containing clones was used to determine the nucleotide sequence coding for the lambda light chain constant (C) region. The deduced amino acid sequence of this cDNA was found in good agreement with a Basilea rabbit C lambda region amino acid sequence previously determined. The nucleotide sequence of the rabbit C lambda-coding region was compared with man, mouse and chicken C lambda sequences and showed 78%, 72% and 66% homology, respectively. Southern blot hybridization analyses of liver DNA from various rabbits were carried out. The comparison of the restriction patterns suggests that a few C lambda-related genes occur in the rabbit genome. In addition, discrete differences in the restriction patterns may exist between rabbits of different genetic backgrounds.     

Isolation and sequence of a cDNA coding for the immunoglobulin mu chain of the sheep.

A sheep cDNA library was screened with a human C mu probe, and the complete nucleotide sequence of a 1923 nt cDNA was determined. It contains sequences corresponding to all the exons (VH, DH, JH, CH1, CH2, CH3 and CH4) characteristic of the immunoglobulin mu heavy chain regions. The deduced amino acid sequence shows a percentage of identical residues in the range 65-45% when compared with the mu chains of various species. The VH region of this clone is clearly related to a group of genes that includes mouse VH36-60 and VHQ52, human VH2, VH4 and VH6 gene families and Xenopus VHII gene families. The constant region shows an unusual repartition of cystein and proline residues at the beginning of the CH2 domain, that may result in a molecule with enhanced stability and reduced flexibility.     

Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe

A cDNA clone complementary to the mRNA encoding human myosin heavy chain has been isolated from a human fetal skeletal muscle cDNA library. A 600 base pair fragment of the inserted human cDNA has been used as probe in the Southern analysis of DNA from panels of rat/human and mouse/human somatic cell hybrids. All the sequences detected by this probe have been mapped to chromosome 17 in the region 17pter → 17p11. There was no evidence for MHC sequences on any other chromosome.     

Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus

Summary.   The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves. Received Jnuary 24, 2000 Accepted August 1, 2000     

Hypermutation in shark immunoglobulin light chain genes results in contiguous substitutions

Among 631 substitutions present in 90 nurse shark immunoglobulin light chain somatic mutants, 338 constitute 2-4 bp stretches of adjacent changes. An absence of mutations in perinatal sequences and the bias for one mutating V gene in adults suggest that the diversification is antigen dependent. The substitutions shared no patterns, and the absence of donor sequences, including from family members, supports the idea that most changes arose from nontemplated mutation. The tandem mutations as a group are distinguished by consistently fewer transition changes and an A bias. We suggest this is one of several pathways of hypermutation diversifying shark antigen-receptor genes--point mutations, tandem mutations, and mutations with a G-C preference--that coevolved with or preceded gene rearrangement.     

Sequence of a rat immunoglobulin gamma 2c heavy chain constant region cDNA: extensive homology to mouse gamma 3

The complete sequence of the rat immunoglobulin gamma 2c heavy chain constant region has been determined by cDNA cloning and by mRNA sequencing. Comparison with other heavy chain genes reveals a high degree of homology (87%) to mouse gamma 3 and suggests that rat and mouse gamma genes separated from a common set of three ancestral genes.     

Extensive allelic sequence variation in the J region of the human immunoglobulin heavy chain gene locus

During the initial stages of B lymphocyte differentiation heavy chain variable (VH), diversity (DH) and joining (JH) gene segments recombine to form a functional heavy chain variable region (VDJ) gene. Evidence for genetic polymorphism of the human JH gene segments has been obtained from mature rearranged VDJ sequences. We conducted an analysis of the published rearranged JH gene sequences and found that the JH alleles present in the two published germ-line JH region sequences were rare (approx. 2%) in the rearranged sequences. As an attempt to explain this discrepancy a 2.5-kb strech of DNA containing all the six heavy chain JH region genes and the most 3' DH gene segment, DHQ52, was amplified by the polymerase chain reaction from 39 individuals and analyzed for restriction fragment length polymorphism. Five new JH region haplotypes were found and sequenced. These new haplotypes contained the coding segment alleles that were frequent in antibody genes. Surprisingly, a high number of interallelic differencies in the non-coding sequence was found between the new and the two previously published haplotypes implying that the haplotypes had been separated early in evolution. In this respect the JH locus resembles HLA loci.     

The role of immunoglobulin heavy chain binding protein: in immunoglobulin transport

More than ten years ago a heavy chain binding protein (BiP) was described which is associated with immunoglobulin heavy chains (HC) within the endoplasmic reticulum (ER) [which is the site of Immunoglobulin (Ig) assembly].Recently, Linda Hendershot and her collegues suggested that BiP might combine with nascent HC as they enter the ER and hold them there until assembly with light chain (LC) occurs. In the absence of LC synthesis or assembly, the HC would remain associated with BiP and would eventually be degraded internally.They now propose a means for BiP to block the transport of unassembled Ig molecules. Transport of protein from ER to the Golgi apparatus seems to be mediated by transport signals inherent to the protein molecule itself. Ig transport signals have been thought to be on the LC because LC can be secreted alone while HC cannot under normal circumstances. When BiP is displaced by LC, completed Ig molecules are transported.They use this model to explain regulated transport of Ig molecules during B-cell development, and suggest that BiP may be post-translationally associated with the nascent chains of other membrane and secretory proteins before folding or subunit assembly.     

Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype

Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity. These antibodies were purified by either immunoabsorption or ion exchange chromatography. The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined. Partial sequence data were also obtained for the two other heavy chains. The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities. The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2. Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities. This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.     

Isolation of a strain-specific Entamoeba histolytica cDNA clone.

Entamoeba histolytica is an intestinal parasite causing significant morbidity and mortality worldwide. More tools are needed to understand the epidemiology and molecular pathogenesis of amebiasis. A cDNA library was constructed by using poly(A)+ RNA isolated from an axenic strain of E. histolytica, HM1:IMSS, which expresses a pathogenic isoenzyme pattern (zymodeme). Differential screening of the library yielded a strain-specific 3' polyadenylated cDNA clone, C2, possessing nine 26-nucleotide tandem repeats. RNA and DNA transfer blot analysis of four axenic strains of E. histolytica possessing the same pathogenic zymodeme revealed that the gene is present and expressed in pathogenic E. histolytica HM1:IMSS and 200:NIH but is not present in pathogenic strains HK-9 and Rahman. In addition, Southern blot analysis using the C2 clone showed heterogeneity of genomic organization between HM1:IMSS and 200:NIH. DNA dot blot hybridization analysis demonstrated that cDNA clone C2 was also able to distinguish axenically cultured E. histolytica strains possessing pathogenic zymodemes from those possessing nonpathogenic zymodemes and could detect as few as 100 amebic trophozoites. We conclude that C2 is a strain-specific E. histolytica cDNA clone that, in conjunction with other E. histolytica-specific probes, could serve as a useful epidemiologic tool.     

Receptor revision of immunoglobulin heavy chain genes in human MALT lymphomas.

BACKGROUND/AIMS: Rearrangement of immunoglobulin gene segments, leading to B cells with functional receptors, is thought to be largely restricted to developing immature B cells in bone marrow. However, accumulating evidence suggests that mature B cells occasionally modify their antigen specificity by VH segment replacements during the germinal centre reaction to enhance antigen affinity, or to overcome self reactive antigen receptors. Although malignant B cells maintain the features of their normal counterparts in most instances, to date, such replacements have not been described for human B cell lymphomas. METHODS: Rearranged immunoglobulin heavy chain genes from two extranodal marginal zone B cell lymphomas were amplified, cloned, and sequenced. Sequences with identical CDR3 regions were selected and aligned to each other and public databases. RESULTS: VH replacements were seen in two extranodal marginal zone B cell lymphomas. In line with the hypothesis that in mature B cells these replacements are associated with active somatic hypermutation, in addition to VH replacement, different mutation patterns were seen in the revised VH portions. In the remaining common 3'-VH regions, these mutations could be used to establish a phylogenetic relation between the sequences, rendering the possibility of artefactual chimaeric polymerase chain reaction products very unlikely. CONCLUSIONS: These results support the view that VH replacements are a further mechanism for reshaping antigen affinity and specificity, and indicate that these receptor modifications are not restricted to normal and reactive germinal centre B cells, but may also occur in close association with the development of malignant B cell lymphomas.     

Amino-acid sequence of squid myosin heavy chain

This work describes the determination of the cDNA sequence encoding the myosin heavy chain (MHC) of the squid, Loligo pealei. To date, the amino-acid sequence of the MHC of calcium-regulated myosins is known only for two closely related species of scallops. We have determined the sequence of the entire coding region of the muscle MHC of squid, a cephalopod, and compared it with the MHC of scallops, which are pelecypods, and to other regulated and non-regulated myosins. Residues present in the MHC of only regulated myosins have been identified. The 6504 base pair (bp) sequence contains an open reading frame of 5805 nucleotides, which encodes 1935 amino acids. The sequence includes 697 bps of 3 untranslated sequence and 2 bps of 5 untranslated sequence. The deduced amino-acid sequence shows the squid MHC to be 72–73% identical and 86–87% similar to the calcium-regulated scallop MHCs cloned previously. In contrast, the squid MHC sequence is only 54–55% identical and 74% similar to skeletal MHCs of non-regulated myosins such as human fast skeletal embryonic and human perinatal skeletal muscle, and 39–40% identical and 60–62% similar to smooth muscle MHC of rabbit uterus muscle and chicken gizzard muscle, respectively. We have also detected two isoforms of the MHC in squid that appear to be spliced variants of a single myosin gene. These isoforms differ in the sequence encoding the surface loop at the nucleotide binding site. Taken together, our data may help to identify more precisely the residues that are crucial in regulated myosins.This revised version was published online in September 2005 with corrections to the Cover Date.     

cDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos

A cDNA was cloned which encoded an immunoglobulin (Ig) light (L) chain of the White Pekin duck. The organization of the variable (V) and constant (C) domains was analyzed by genomic Southern blotting. The duck L chain gene has a similar chromosomal organization to that of the chicken, with a single λ-like C region and multiple V_L hybridizing elements. The amino acid sequence of the V_L region of the White Pekin duck L chain showed 88% identity with the Muscovy duck and 87% identity with the chicken, the J_L region showed 92% identity with these species, and the C_L region showed 88% identity with Muscovy duck and 66% with chicken. The constraints imposed by the gene-conversion mechanism of generating antibody diversity might account for the similarities of the avian V region sequences.