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R P Perry

Publications and source records attributed to R P Perry.

At least 73 records · Page 4Linked to original sources
PubMed1983

Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma.

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.

Animals↗
PubMed1983

Methylation status and DNase I sensitivity of immunoglobulin genes: changes associated with rearrangement.

Immunoglobulin V kappa genes are transcriptionally silent in their germline context and become transcriptionally active upon fusion to the J kappa-C kappa region (kappa locus). To elucidate the role of chromosomal structure in this regulatory phenomenon we have investigated the DNase I sensitivity and methylation status of the kappa locus and selected V kappa genes in a variety of alleles exhibiting different rearrangement configurations and different levels of transcriptional activity. Our findings indicate that the kappa locus in either germline or rearranged contexts maintains a distinctive DNase I-sensitive, hypomethylated structure in plasmacytomas and hybridomas, irrespective of its level of transcriptional activity. In contrast, the germline V kappa genes are in less accessible regions of chromatin and more highly methylated regions of DNA. Upon fusion to the kappa locus, V kappa genes become DNase I-sensitive and hypomethylated. This effect extends several kilobases upstream of the transcriptional initiation site but does not extend to the adjacent V kappa gene or to the identical V kappa allele on the other chromosome, indicating that the structural alteration is a localized cis-acting phenomenon.

Animals↗
PubMed1983

Characterization of productive and sterile transcripts from the immunoglobulin heavy-chain locus: processing of micron and muS mRNA.

An analysis of the sizes and sequence content of nuclear RNA transcripts of the heavy-chain locus in two B-cell lymphomas, 70Z/3 and 38C-13, and in selected hybridoma derivatives of 38C has led to the identification of two distinct precursors of the mRNAs encoding the membrane and secretory forms of mu chain. These precursors, termed Pm1 and Ps1, extend from a common 5' terminus (presumably the cap site) to alternative polyadenylation sites located 3' of the membrane and secretory tailpieces, Pm1 and Ps1 are present in similar amounts in lymphomas, indicating roughly equivalent usage of the two polyadenylation sites, whereas Ps1 much greater than Pm1 in hybridomas, indicating that mature plasma cells produce a trans-acting factor which enhances cleavage at the proximal (muS) site. The lymphomas also synthesize several nonproductive or sterile mu (Smu) transcripts from the second H allele. One class of sterile mu transcripts appears to be initiated about 1 kilobase downstream from the JH4 element. In 70Z, in which the nonproductive H allele has undergone a D1J2 fusion, another initiation site was located about 0.3 kilobase upstream of the D1 element. The sterile mu transcripts exhibit the same regulated termination at alternative polyadenylation sites as the mu mRNA precursors, although their rate of production is not necessarily coupled to that of the productive allele. This analysis has also defined probable processing pathways for productive and sterile components in which there is a 5' leads to 3' order for the excision of the large introns.

Animals↗
PubMed1982

Functional significance and evolutionary development of the 5'-terminal regions of immunoglobulin variable-region genes.

The 5'-terminal sequence of the kappa light-chain gene expressed in MPC11 plasmacytoma cells was definitively determined by analysis of both the kappa mRNA and the gene from which it is transcribed. The distance between the cap site and the translation initiation codon is only 3 nucleotides, and in a minor variant only 2 nucleotides, considerably less than that found in other species of nucleus-derived mRNA. S1 nuclease protection experiments with MPC11 nuclear RNA indicate that the cap sites are coincident with the transcriptional start sites, suggesting that the 5'-terminal heterogeneity is caused by imprecision in transcriptional initiation. A comparison of the 5'-terminal structure of the MPC11 V kappa gene with that of several other V genes indicates that the length of the first exon, which is composed of the 5' untranslated region which is composed of the 5' untranslated region and a sequence encoding most of the signal peptide, is highly conserved. Within this set of examples, the 5' untranslated region varies from 3 to 33 nucleotides, and the signal-peptide-coding block from 46 to 76 nucleotides. This analysis has also provided insight into the genetic origins for two anomalous properties of the MPC11 light chain.

Base Sequence↗
PubMed1982

DNA between variable and joining gene segments of immunoglobulin kappa light chain is frequently retained in cells that rearrange the kappa locus.

A systematic analysis of the fate of the DNA between kappa chain variable (V kappa) and joining (J kappa) genes in cells that have rearranged kappa loci was carried out. The DNA from a variety of kappa-producing plasmacytomas, lambda-producing hybridomas, and kappa-expressing lymphocytes was digested, fractionated by size, and analyzed with two probes containing sequences 5' of J kappa. In 13 of 28 plasmacytomas examined the rearrangement of V kappa and J kappa appears to be accompanied by loss of DNA upstream of J kappa. However, in the rest of the plasmacytomas one or more upstream sequences are retained in a new context. In 9 of 12 lambda-producing hybridomas (which frequently rearrange both kappa loci) one or more upstream segments were detected. These unique fragments were probably generated by a recombination event near or at the J kappa region. The extent to which the region between V and J is maintained in kappa-expression lymphocytes was also measured. Most (76%) of the region upstream of J kappa is retained in the population, even though 68% of the kappa loci are rearranged. In order to explain how these upstream elements occur in some, but not all, cell lines, and the significant occurrence in the lymphocyte population, we propose a model in which a step in V--J joining involves mitotic recombination by unequal sister chromatid exchange.

Alleles↗
PubMed1982

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts.

When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.

Animals↗
PubMed1981

Transcriptional regulation of immunoglobulin V genes.

The relative transcriptional activity of rearranged and unrearranged (germline) VK genes in secreting plasmacytoma cells was assessed by two independent methods. Measurements of V sequence abundance by hybridization kinetic (Rot) analysis indicated that the steady state content of transcripts from a rearranged VK gene is at least 16,000-fold greater than that from an unrearranged VK gene. Direct measurements of transcriptional activity in isolated nuclei indicated that this difference is due, in large part to a difference in transcription rate. Since the primary sequences of V genes and their 5' flanking regions are not altered during rearrangement, these results suggest that VK gene transcription might be controlled by elements on the 3' side of the VK genes or at the CK locus, perhaps via an influence on chromatin structure.

Animals↗
PubMed1981

Mammals have multiple genes for individual ribosomal proteins.

The reiteration frequency of the genes that encode the structural proteins of the mammalian ribosome was studied with a set of cloned cDNA probes containing several different mouse r-protein mRNA sequences. Results from a reassociation kinetics analysis, Southern blotting experiments and gene cloning studies collectively indicate that each individual r-protein species is represented by multiple genes in mammals. Among the examples studied, the multiplicity of mouse r-protein genes varied from about 7 to 20, a striking contrast to the low copy numbers observed in less evolutionarily advanced eucaryotes. The multiplicity of individual r-protein genes in humans and rodents is similar.

Animals↗
PubMed1981

Chromosomal distribution of ribosomal protein genes in the mouse.

The chromosomal distributions of five families of mouse r-protein genes (S16, L18, L19, L30 and L32/33) were studied by Southern blot analysis of DNa from a panel of mouse-hamster hybrid cells containing various complements of mouse chromosomes. Our results indicated that members of a particular family are often located on more than one chromosome, that extensive clustering of many r-protein gene families on a few chromosomes is unlikely, and that there is no obligatory linkage of r-protein and rRNA genes.

Animals↗
PubMed1981

Transcription of the unrearranged mouse C kappa locus: sequence of the initiation region and comparison of activity with a rearranged V kappa-C kappa gene.

In cells of the B-lymphocyte lineage, 8.4 kb transcripts are constitutively produced from unrearranged kappa constant region (kappa 0) loci. To help elucidate the molecular basis of this phenomenon, we have determined the nucleotide sequence surrounding the site of transcriptional initiation. The kappa 0 transcripts are initiated within a unique Eco RI fragment located about 8 kb upstream from the C kappa gene. The start site is about 36 nucleotides downstream from a Hogness consensus sequence (TGTAAAT) and nearly 200 nucleotides upstream from a sequence that is similar to those encoding the signal peptides of kappa light chains. These features, which are usually found in the 5' flanking regions of kappa variable region genes, suggest that the kappa 0 initiation sequence may be an evolutionary relic of some common ancestral 5' element. In contrast, there is no discernible V kappa-encoding element in 780 nucleotides of sequence downstream from the initiation site. From pulse-chase-labeling experiments with a pre-B-cell hybridoma line and direct measurements of transcriptional activity in isolated nuclei, we have estimated that the rate of transcription of the kappa 0 locus is significantly lower than that of a rearranged V kappa-C kappa gene. This result, together with the fact that unrearranged V kappa genes are transcriptionally silent, suggests that structural features of both the V kappa and C kappa loci contribute to the overall transcriptional efficiency of a rearranged V kappa-C kappa gene. The 8.4 kb transcripts are not processed into any stable RNA products, despite the fact that they contain some apparently normal splice junctions; rather, they are degraded within the nucleus at about half the rate with which a kappa mRNA precursor is processed. Conceivably, the transcriptional activity of the kappa 0 locus might be a prerequisite for its recombinatorial activity.

Amino Acid Sequence↗
PubMed1981

Organization and expression of immunoglobulin genes in fetal liver hybridomas.

The organization and expression of immunoglobulin genes were studied in a series of six hybridomas derived from the fusion of a nonproducing myeloma cell with cells from mouse fetal liver. These hybridomas, which exhibit several phenotypic characteristics of immature B lymphocytes, all have productively rearranged mu heavy chain genes and produce both the membrane and secreted forms of mu mRNA in a ratio of about 1:10. Significantly, none of the hybridomas has an unrearranged (germ line) allelic mu gene. Examination of the kappa light chain genes revealed that all six of the hybridomas contain unrearranged kappa loci and produce 8.4-kilobase transcripts containing kappa constant region sequences. None of the five hybridomas that exhibit a mu-only phenotype contains a rearranged kappa gene other than that derived from the myeloma parent. One hybridoma, which actively secretes kappa immunoglobulin, contains a rearranged kappa gene of fetal liver origin and synthesizes a distinctive kappa mRNA precursor in addition to the 8.4-kilobase transcript. These results demonstrate that rearrangement of heavy chain immunoglobulin genes normally occurs prior to that of light chain genes and further indicate that the transcriptional competence of the kappa constant region locus is established prior to the time of its rearrangement.

Animals↗
PubMed1980

Rearrangement of immunoglobulin heavy chain genes during B-lymphocyte development as revealed by studies of mouse plasmacytoma cells.

We have used Southern's blotting technique to determine the extent to which the genes encoding the constant (C) regions of mu, alpha, gamma(1), and gamma(2b) immunoglobulin heavy (H) chains are altered in number and context from their germline (embryo) state in a series of 14 plasmacytomas expressing various H chain classes. In the three mu chain-producing plasmacytomas studied there was no evidence of rearrangement of C(H) genes other than C(mu). In contrast, rearrangement and deletion of nonexpressed C(H) genes was frequent in plasmacytomas that produce gamma or alpha chains. The observed pattern of deletions is consistent with the idea that the ontogenetic switch in H chain class requires C(H) gene deletion. Frequently, though not always, such deletions as well as other types of rearrangement occur in both allelic loci. Particularly noteworthy are three gamma(2a)-expressing tumors in which C(alpha) gene rearrangement is evident in both alleles. We incorporate these observations into a probabilistic model of B cell development: in the first phase, deletions may occur between the C(mu) gene and the variable (V(H)) gene array, which result in the formation of a productive fused V(H)-C(mu) gene. The cell may then enter a second phase, which allows deletions within the C(H) gene arrays of both homologous chromosomes. Some deletions juxtapose the expressed V(H) gene with a second C(H) gene and result in a H chain class switch; others delete or alter the context of C(H) genes without changing the phenotype of the cell. We predict that switching can be both a single-step and a multi-step process, and that in the latter case those rearrangements that do not result in a switch may be physiologically significant in that they may limit the options of further switching.

Animals↗
PubMed1980

Transcription of mouse kappa chain genes: implications for allelic exclusion.

The nuclear RNA from a large variety of kappa-producing plasmacytomas was size fractionated and analyzed with a series of cloned probes representing sequences encoding variable (V), joining (J), and constant (C) regions and selected intervening sequences. All of the plasmacytomas produce a nuclear RNA component that contains V kappa and C kappa sequences as well as the intervening sequence between J kappa and C kappa, and that has a distinctive size depending on which of the four J kappa segments is expressed (i.e., is present in the secreted kappa chain). These RNAs are the precursors of kappa mRNAs, which are transcribed from productively rearranged C kappa genes. Half of the plasmacytomas examined produce, in addition to a kappa mRNA precursor, a discrete component of about 8.4 kilobases that contains C kappa and upstream flanking sequences but lacks the expressed V region sequence. The ability to produce this component is always associated with the persistence in the tumor genome of an unrearranged (germline) J kappa-C kappa region. In tumors rearranged at both kappa loci the nonproductive allele is either transcriptionally silent or, in a minority of cases, transcribed and processed into a "fragment" mRNA lacking V region sequences. These results reveal that allelic exclusion can be effected at several levels of gene expression. They also provide some insight into the relative contributions of the V and C gene elements to this expression.

Alleles↗