Structure of a germline rabbit immunoglobulin V kappa-region gene: implications for rabbit V kappa-J kappa recombination

Rabbit kappa-immunoglobulin chains exhibit diversity in the number of amino acids between the invariant residues Cys 88 and Phe 98; this length diversity is formally similar to that found in the human and mouse heavy chain systems, in which it results from interposition of the D element between V and J. To explore the molecular basis for this length diversity in rabbit kappa-chains we have determined the nucleotide sequence of a rabbit germline V kappa immunoglobulin gene. The spacing between the 7-mer and 9-mer signal elements of this gene suggest that it could recombine with J kappa without a D element. We discuss alternative explanations for the length diversity of rabbit kappa-chains.     

Subgroup IV of human immunoglobulin K light chains is encoded by a single germline gene.

The series of studies on the human K light chain genes of the various subgroups is concluded by this report on the isolation and nucleotide sequence determination of a functional VKIV gene (abbreviations ref. 1) and its germline counterpart. The rearranged gene which stems from a lymphoid cell line and the germline gene differ in four nucleotides which can be attributed to somatic mutations; three of the mutations are clustered in CDR3. The germline gene regions of two unrelated individuals were identical over a stretch of 1267 bp. By hybridization experiments it is shown that the human K locus contains only one VKIV gene. In 16 lymphoid cell lines studied here, the VKIV gene is frequently deleted or aberrantly rearranged which may be a consequence of peculiarities of its function and/or its structural organization.     

Nucleotide sequence of a mouse kappa light chain cDNA cloned in a bacterial plasmid.

A mouse MOPC21 cDNA previously cloned in plasmid pMB9(Higuchi $\text{et}\text{al.}$, Proc. Natl. Acad. Sci. 73 (1976) 2136–2140; Wall $\text{et}\text{al.}$, Nucleic Acid Res. 5 (1978) 3113–3128) and is designated pL21-3 has been extensively characterized. Cleavage of pL21-3 with Hpall has shown the insert to be 910 basepairs long, consistent with the length of the entire variable and constant regions and the untranslated regions. Digestion of pL21-3 with various restriction endonucleases has established that the insert sequence starts from parts of the 5′ leader region and extends downstream to include the untranslated 3′ terminus. 131 nucleotides in the variable region corresponding to amino acids 49–91 have been determined.     

Brain-specific expression of MAP2 detected using a cloned cDNA probe

We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.     

The human V kappa locus. Characterization of extended immunoglobulin gene regions by cosmid cloning

As part of the ongoing work in our laboratory on the structural organization of the human V kappa locus we screened cosmid libraries with V kappa gene probes and obtained numerous V kappa gene-containing cosmid clones. Several genomic regions of the V kappa locus were reconstructed from overlapping cosmid inserts and were extended by one step of chromosomal walking. The regions that are called Wa, Wb, Oa, Ob and Ob' comprise about 370 kb (10(3) bases) of DNA and contain 24 V kappa genes and pseudogenes. The V kappa genes belong to the three dominant subgroups (V kappa I, V kappa II, V kappa III) and are arranged to form mixed clusters with members of the different subgroups being intermingled with each other. The distances between the genes range from 1 to 15 kb. Three genes of the Wa and Wb regions that were sequenced turned out to be pseudogenes. Terminal parts of the regions Wa and Ob that do not contain V kappa genes of one of the known subgroups may represent extended spacer regions within the V kappa locus. Wa and Wb are duplicated regions located at different positions of the locus. Region Wb was found to comprise inversely repeated sections of at least 14 kb each that contain V kappa genes oriented in opposite polarity. This finding is consistent with inversion-deletion models of V-J joining; it also shows that the V kappa locus contains not only unique and duplicated but also triplicated parts. The data on the W and O regions are discussed together with those on the L regions and on other regions established in our laboratory. Although the picture of the human V kappa locus with, to date, about 70 different non-allelic V kappa genes is still incomplete, some general features with respect to the organization of the genes and the limited duplication of genomic regions have emerged.     

Interstrain conservation of the murine GAT-specific antibody V kappa repertoire as analyzed at the germline gene level

A cDNA library was constructed in pBR322 from mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) monoclonal antibody kappa chain. Two cDNA clones were extensively characterized. One, L XI 62, was derived from an aberrant V kappa-J kappa rearrangement which resulted in a frame-shift at position 96, leading to a stop codon at the very beginning of the constant region. The second, L XIX 27, 1150 bp long, was unequivocally assigned to a GAT-specific kappa chain, by comparison of its nucleotide sequence with the previously determined NH2-terminal amino acid sequence of the isolated kappa chain. A specific probe, containing the leader and most of the V kappa gene-encoded region, was prepared from this clone and hybridized to EcoRI and BamHI restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains. Under stringent conditions, similar patterns were observed for all three strains, and consisted of a small number of bands (3-5). Under nonstringent conditions, patterns were again very similar when the different strains were compared, although 15-20 bands could be identified. These observations support the hypothesis that the GAT-specific kappa chains found in antibodies expressing the public CGAT idiotypes are encoded by a very small number of germline genes. This V kappa repertoire seems extremely conserved between the three strains that were analyzed, an observation which correlates with the interstrain conservation of these public idiotypic specificities.     

Nucleotide sequence of cloned cDNA of human apolipoprotein A-I.

ApoA-I is the major human HDL apoprotein. By oligonucleotide hybridization, we have isolated 5 dscDNA clones to human hepatic apo A-I mRNA. One of these clones (pA1-3) was completely sequenced. It has 878 bp plus a poly A tail of 48 and includes all the coding and 3'-untranslated regions of the mRNA and part of the 5'-untranslated region. It predicts a peptide sequence of 267 amino acids (including the 24 amino acid prepropeptides) which is very similar to the sequence reported by Brewer et al., (1978) Biochem. Biophys. Res. Commun. 80:623-630. The predicted signal peptide sequence is highly homologous to the rat apoA-I signal peptide. There is no evidence for any internally repeated segments in apoA-I either at the amino acid or at the DNA level. Using pA1-3 as a probe, we have detected on Northern gels apo A-I mRNA sequences of approximately 1100 nucleotides in human hepatic and baboon hepatic and intestinal RNAs, but not in RNAs from baboon skeletal muscle, kidney or spleen. The demonstration of apo A-I mRNA sequences in specific organs is important to our concept of "reverse cholesterol transport".     

The nucleotide sequence of a cloned human leukocyte interferon cDNA

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.     

The sequence of human parathymosin deduced from a cloned human kidney cDNA

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine. Analysis of the 5' flanking sequence excluded the presence of a hydrophobic signal peptide in the translated sequence. It may therefore be concluded that parathymosin, like prothymosin alpha, is synthesized without formation of a large precursor polypeptide. Comparison of the deduced amino acid sequence with the known primary structure of rat and bovine parathymosins shows that the primary structure of parathymosin is highly conserved among these species.     

The structural repertoire of the human V kappa domain.

In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa-J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed.     

A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina.

The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.     

Characterization of a group of transposed human V kappa genes

A genomic region with three V kappa pseudogenes which has been transposed to chromosome 22 is characterized by detailed restriction mapping. A number of subclones are described one of which proved useful to establish an allelic restriction fragment length polymorphism (RFLP) in the region. Allelic and duplication-derived restriction site differences in cosmid clones are discussed with respect to possible problems in genomic walking experiments.