The variable genes and gene families of the mouse immunoglobulin kappa locus.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur.

The human immunoglobulin kappa locus on yeast artificial chromosomes (YACs).

The human immunoglobulin kappa locus is a duplicated structure. Contigs of 600 kb with 40 Vkappa genes and 440 kb with 36 Vkappa genes had been established for the Ckappa proximal (p) and distal (d) copies, respectively. In addition the human genome contains more than 24 dispersed Vkappa genes, called orphons.

Searching for non-V kappa transcripts from the human immunoglobulin kappa locus.

The 76 V kappa (variable) gene segments of the human immunoglobulin kappa locus (Ig kappa) were cloned in two contigs. Within each contig the distances between the genes range from a few hundred bp to 31 kb. We have now studied the question of whether transcripts are produced from intergenic regions, from regions near the kappa locus or from V kappa orphon regions.

A potentially functional V kappa gene at a distance of 1.5 Mb from the immunoglobulin kappa locus.

An amplicon that is highly homologous to a part of the human kappa locus was found at the 3' or telomeric side of C kappa at a distance of about 1.5 Mb. From analysis of sequence divergence, it is concluded that the amplicon was formed after the duplication of the kappa locus, which may have taken place about 1 million years ago.

The human immunoglobulin kappa locus: pseudogenes, unique and repetitive sequences.

The human kappa locus contains 25 pseudogenes. After seven of them were described earlier the structures of the remaining 18 are reported now, thus completing the description of all human V kappa genes and pseudogenes. Most of the pseudogenes carry several defects each.

The V kappa genes of the L regions and the repertoire of V kappa gene sequences in the human germ line.

Only 14 of the 25 V kappa genes and pseudogenes had been found before as parts of the L regions. The cloning and linking described in the accompanying report allowed us now to assign to Lp or Ld some V kappa genes which had been found before on scattered clones. In addition the sequences of several still unknown genes are reported here, thus completing the publication of the V kappa genes of the kappa locus as far as they are potentially functional or have only one or two 1-bp defects.

Of orphons and UHOs. Delimitation of the germline repertoire of human immunoglobulin kappa genes.

Two problems in defining the germline repertoire of immunoglobulin kappa genes were investigated. One concerns putative transposed V kappa genes (orphons), the other one weak hybridization signals which may or may not turn out to be V kappa genes (UHOs). It was shown by sequencing that the three V kappa genes Z2, Z3 and Z4 are very closely related to the Z1 and V118 genes and to two other genes which had been localized on chromosomes 1 and 22, i.

The human immunoglobulin kappa locus. Characterization of the duplicated A regions.

The central regions of the kappa locus, the so-called A regions, have been fully characterized on cosmid and phage lambda clones. The regions, which are parts of the C kappa-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 V kappa genes and pseudogenes. The A regions have been linked on their 5' sides to the O regions and on their 3' sides to the L regions.

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous.

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies.

The human immunoglobulin kappa locus. Characterization of the duplicated O regions.

Two large regions of the human immunoglobulin kappa locus, the so-called O regions, have been characterized on cosmid and phage lambda clones. The two regions are very similar but not identical duplicates belonging to the C kappa proximal (p) and the distal (d) copies of the kappa locus. The Op and Od regions comprise contigs of 90 and 120 kb, respectively, and contain 20 V kappa genes and pseudogenes which have been sequenced.

A human immunoglobulin kappa orphon without sequence defects may be the product of a pericentric inversion.

The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization.

Evolution of a group of transposed human V kappa genes.

Eleven V kappa genes within a genomic region which has been transposed from the short to the long arm of chromosome 2 have been characterized by sequence analyses. Nine of the analysed genes exist within the genome in three highly homologous copies each. Sequence comparisons of the triplicated genes make it very likely that the three copies of a given gene were produced at different times during evolution.

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region.

To characterize the L chain V region repertoire of IgG anti-Haemophilus influenzae type b capsular polysaccharide (Hib-PS) antibodies, clonal antibodies were purified from immune serum and internal amino acid sequences of VKII anti-Hib-PS L chains obtained. We examined VKII L chains because it is the most common VL family expressed in the anti-Hib-PS response. Comparison of VKII amino acid sequences, including the entire CDR2 and CDR3 regions, of five anti-Hib-PS clonal antibodies from four unrelated individuals revealed complete identity with the exception of a single CDR3 residue from one antibody.

The Z family, a group of transposed human immunoglobulin V kappa genes.

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb.

Two unusual human immunoglobulin V kappa genes.

The V kappa genes A10 and A14 which have been previously localized within the human kappa locus were analysed now. A10 hybridizes under stringent conditions only weakly or not at all to probes characteristic for the four V kappa subgroups. According to their DNA sequences and the derived amino-acid sequences A10 and A14 do not fit well into the subgroup classification.

The J kappa proximal region of the human K locus contains three uncommon V kappa genes which are arranged in opposite transcriptional polarities.

The structure of one of the V kappa gene-containing regions of the locus coding for the human immunoglobulin light chains of the kappa type is described. This so-called B region contains three genes: B1, B2 and B3. According to its sequence B1 is a pseudogene which does not fit well into the present subgroup classification.

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.

Catalytical mechanism of the phenylalanyl-tRNA synthetase from yeast. Reactivity of ATP in the absence of phenylalanine.

Phenylalanyl-tRNA synthetase catalyses an AMP-ATP exchange under conditions where no aminoacylation of tRNA occurs. A plausible explanation for this reaction had not been given so far. The results of the present investigation provide evidence for the following interpretation.

No arginyl adenylate is detectable as an intermediate in the aminoacylation of tRNAArg.

On the supposition that aminoacyl adenylate is a necessary intermediate in the reactions catalyzed by aminoacyl-tRNA synthetases, six possible reactions requiring this intermediate were tested. With arginyl tRNA synthetase from brewer's yeast they were all negative and with phenylalanyl-tRNA synthetase they were all positive. Therefore, no evidence for the formation of arginyl adenylate could be provided.

Arginyl-tRNA synthetase from brewer's yeast. Purification, properties, and steady-state mechanism.

tRNAArg and arginyl-tRNA synthetase have been purified to homogeneity from brewer's yeast by chromatographic methods. Arginyl-tRNA synthetase is a monomeric enzyme with a molecular weight of 72000. Two active forms of the enzyme can be found, they are interconvertible.

Interference of ligands on the phenylalanyl-tRNA synthetase from yeast.

The present paper reports a study of the mutual interactions between the substrates, the intermediate, and the products of the aminoacylation reaction, when bound to the phenylalanyl-tRNA synthetase from yeast. The following conclusions can be drawn. a) tRNAPhe displaces Phe-tRNAPhe from the synthetase by lowering the affinity of the enzyme for the aminoacylated tRNA.

Analysis of the steady-state mechanism of the aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase from yeast.

The steady-state mechanism of the aminoacylation of tRNAPhe by the corresponding synthetase from yeast has been investigated in detail by kinetic experiments. It was found that there are two alternative mechanisms: one favoured at low tRNA concentrations and the other at high tRNA concentrations. ATP and Phe are bound randomly to the enzyme.