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U Storb

Publications and source records attributed to U Storb.

At least 55 records · Page 3Linked to original sources
PubMed1983

Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.

Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.

Animals↗
PubMed1982

Evolution of mouse immunoglobulin lambda genes.

The mouse has four C lambda and two V lambda genes. We have isolated Charon 4A clones that contain all six lambda genes from a BALB/c germ-line library. We present here the DNA sequences of the C lambda 2, C lambda 3, and C lambda 4 genes and also correct what are apparently errors in previous reports of C lambda 1 protein and DNA sequences. In addition, we have analyzed cloned DNAs by restriction mapping and electron microscopy to determine the relationships among the various lambda genes. By heteroduplex analysis, two gene clusters containing JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 show homology extending from the J regions 5' of C lambda 3/C lambda 2 to just 3' of C lambda 1/C lambda 4. Other than the region between the genes, very little homology exists in the C lambda flanking regions. In contrast, V lambda 1 and V lambda 2 genes show considerable homology extending into the 5' flanking regions. Large inverted repeats are found in the 5' flanking regions of V lambda 1 and C lambda 3, as well as in the 3' flanking regions of both C lambda gene clusters. DNA sequence divergences between the C lambda genes indicate that an ancestral JC lambda x--JC lambda g gene cluster arose at about the time of the first mammals by duplication of a primordial JC lambda gene. The data further suggest that the JC lambda x--JC lambda gene cluster duplicated after the speciation of mouse and man and subsequently diverged into the present day JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 gene clusters. C lambda 4, a pseudogene, became inactive at about the time of duplication of the ancestral JC lambda x--JC lambda y cluster. Comparison of DNA sequence divergence between the V lambda 1 and V lambda 2 genes demonstrates an anomaly. The percentage of amino acid replacement changes is approximately the same for V lambda 1/V lambda 2 as for C lambda 3/C lambda 2, implying that the ancestral V lambda gene was duplicated at the same time, and possibly together with, the JC lambda x--JC lambda y cluster. However, there are fewer silent changes than amino acid replacement changes between the V lambda 1/V lambda 2 genes, suggesting either that a selective pressure acted on the silent sites or that V lambda genes have only recently been duplicated. We also consider the possibility of a gene conversion event subsequent ot a more ancient duplication.

Animals↗
PubMed1981

Mapping of immunoglobulin variable region genes: relationship to the 'deletion' model of immunoglobulin gene rearrangement.

Five families of variable region genes of mouse kappa chains were analyzed by Southern blot hybridization to determine their relative chromosomal map positions. Map positions were deduced by Vk gene deletion from antibody-producing cells expressing upstream Vk genes and retention in cells expressing downstream genes. The Vk regions expressed in the myelomas M0PC167, MPC11, M0PC21 and ABPC20 are members of Vk families exhibiting one, three, six and six major germline hybridization bands respectively. The gene order of the five families in germline DNA was found to be VM167-VM11-(VM21, VA20)-VABE8-Jk-Ck. As expected in a deletion model of immunoglobulin gene rearrangement, a sequence located just 5' of J1 in germline DNA was found to be absent from some antibody producing cells which had not retained any germline Ck genes. However, other cell lines contained this sequence in rearranged contexts, suggesting that any deletion model of immunoglobulin V-J joining, as well as V gene mapping, must take into account the possibilities of stepwise rearrangements and reintegration of "deleted" DNA.

Animals↗
PubMed1981

Immunoglobulin mu- and gamma-ribonucleic acid sequences in thymocytes and splenocytes from normal and hyperimmune mice.

The immunoglobulin heavy-chain ribonucleic acid (RNA) repertoire of mouse thymocytes was examined. Previously, this laboratory reported immunoglobulin alpha-chain RNA sequences in mouse thymocytes [Near, R. I., & Storb, U. (1979 Biochemistry, 18, 964]. We have extended these studies to encompass mu, gamma 2b, and gamma 1 heavy-chain RNA sequences, mu-, gamma 2b-, and gamma 1-messenger RNAs (mRNAs) were purified from myelomas to 45, 22, and 54% purity, respectively. Each of these mRNAs faithfully translated into the appropriate immunoprecipitable protein in a reticulocyte lysate translation system. The gamma 1-mRNA translated into two major immunoprecipitable products of about 52 500 and 51 000 daltons while mu- and gamma 2b-mRNAs yielded only a single major protein. Complementary deoxyribonucleic acids (cDNAs) prepared from the mRNAs were used as hybridization probes and revealed the presence of about 70 mu-RNA sequences per average thymocyte as determined by hybridization kinetics, while gamma 1 and gamma 2b sequences were at the limits of detection. The mu-RNA sequences are present in the cytoplasm and are greater than 50% polyadenylated. Upon hyperimmunization of mice with sheep red blood cells, gamma 1-RNA in splenocytes increased by about 100-fold while only slightly increasing in thymocytes. mu and gamma 2b increased 2-3-fold in splenocytes and only slightly in thymocytes. The results argue against RNA sequences appearing in thymocytes due to contamination with peripheral confirmed with cloned cDNA probes. Thymocyte RNA analyzed by Northern blots displayed bands of the same size as those in splenocyte RNA or in purified mRNA when hybridized to mu, gamma 2b and alpha cloned probes. Also, K light-chain RNAs of the same size were found in spleen and thymus by using a cloned K-DNA probe. The results are consistent with the thymus containing mu-, alpha-, and K- and small amounts of gamma 1- or gamma 2b-RNAs coding for heavy- and light-chain-like proteins which may play a role in T-cell function.

Animals↗
PubMed1981

Misalignment of V and J gene segments resulting in a nonfunctional immunoglobulin gene.

The myeloma variant NS-1n has lost the functional immunoglobulin kappa gene which is present in its parent, myeloma MOPC-21. The variant retains a nonfunctional rearranged gene, M.21N, which undergoes RNA transcription and processing to yield a mature size kmRNA. This kRNA, however, is not translated into kappa polypeptide chains. The nonfunctional gene was cloned into Charon 4A to determine the basis for its inactivity. Nucleotide sequence analysis of a DNA fragment overlapping the V-J recombination site in the M.21N gene indicated that a misalignment had taken place during somatic recombination. This misalignment results in a deletion of four nucleotides at the 3' end of the V gene and, thus, a translational reading frame shift. In other respects the M.21n V gene, which corresponds to a different VK subgroup than the functional gene of MOPC-21, appears normal.

Base Sequence↗
PubMed1981

Rearranged and germline immunoglobulin kappa genes: different states of DNase I sensitivity of constant kappa genes in immunocompetent and nonimmune cells.

The rearrangement of a variable (V) and a constant (C) gene appears to be a necessary prerequisite for immunoglobulin gene expression. Multiple different rearranged kappa genes were found in several mouse myelomas, although these cells produce only one type of kappa chain [Wilson, R., Miller, J., & Storb, U. (1979) Biochemistry 18, 5013--5021]. It is therefore of interest to understand how only one allele within a lymphoid cell becomes expressed, while the other allele remains nonfunctional ("allelic exclusion"). We have studied the chromatin conformation of kappa genes by making use of the preferential digestion of potentially active genes by DNase I described, for example, for globin genes [Weintraub, H., & Groudine, M. (1976) Science (Washington, D.C.) 193, 848--856]. The DNase I sensitivity of kappa genes in myeloma tumors, in a B cell lymphoma, and in liver was determined by hybridization with DNA on Southern blots. It was found that rearranged C kappa genes are DNase I sensitive in myelomas in which several kappa genes are rearranged, regardless of whether the rearranged genes code for the kappa chains synthesized by the cell. Furthermore, the C kappa gene in germline configuration is also DNase I sensitive in a B cell lymphoma; i.e., it is in the same chromatin state as the rearranged C kappa gene which probably codes for the kappa chains produced by the cell. The altered chromatin state appears to be localized: V kappa genes in germline context are not DNase I sensitive in myeloma or B lymphoma cells while C kappa genes present in a kappa gene cluster on the same chromosomes are sensitive. When rearranged, however, the V kappa genes are as sensitive to DNase I as are rearranged C kappa genes. V lambda and C lambda genes are not DNase I sensitive in kappa myelomas. Thus, commitment to kappa gene expression is apparently correlated with a chromatin conformation which confers increased DNase I sensitivity to the DNA in the vicinity of all C kappa genes in the cell. "Allelic exclusion" does not operate on the level of chromatin conformation which can be detected by altered DNase I sensitivity.

Animals↗
PubMed1981

Somatic mutation of immunoglobulin light-chain variable-region genes.

A single germline immunoglobulin kappa-variable-region gene, VK167, is rearranged and expressed in two myelomas, MOPC167 and MOPC511. Only this single germline gene displays close homology to the expressed genes. Neither of the rearranged, functional genes, however, has a nucleotide sequence that is identical to the germline VK167 gene. Both active genes display several single-base-pair mutations with respect to the germline sequence. The nucleotide sequence data predict the alteration of a restriction-enzyme-recognition site within the VK167 gene between germline cells and cells producing the MOPC167 light-chain protein. Based on this restriction-site alteration, Southern blot analysis proves unambiguously that no gene present in the germline BALB/c mouse genome contains the exact VK167 nucleotide sequence found in cells committed to MOPC167 antibody production. Instead the alterations found in the expressed MOPC167 and MOPC511 V-region genes have apparently arisen by a process of somatic mutation during cellular differentiation. Since nucleotide alterations are found in framework and hypervariable portions of the variable region, the mechanism of somatic mutation is not limited to hypervariable sequences. In addition, Southern blot hybridization indicates that the observed mutations did not arise by recombinational events, but are single-base-pair substitutions. Based on the distribution of mutations that have been found in expressed immunoglobulin variable-region genes, a model that links the introduction of somatic mutations to DNA replication during the V-J joining event is proposed.

Amino Acid Sequence↗
PubMed1981

Physical linkage of the constant region genes for immunoglobulins lambda I and lambda III.

During differentiation from a stem cell to an antibody-secreting cell, the immunoglobulin genes within a B cell undergo a rearrangement that juxtaposes a variable region gene to a constant region gene. To analyze the genetic organization of an immunoglobulin gene family in nonrearranged, germ-line DNA, we have constructed a recombinant DNA library from randomly cleaved mouse kidney DNA fragments. From this library, we have isolated three overlapping recombinant clones containing the constant region gene for lambda I light chains (C lambda I). These clones spanned 24.9 kilobases of mouse DNA and contained no variable region sequences. Hybridization of these clones with lambda II cDNA demonstrated the presence of an additional constant region gene and a joining region 3.2 kilobases 5' of C lambda I. This gene was tentatively identified as C lambda III by the absence of an Ava I endonuclease site, which is present within C lambda II. The C lambda III amino acid sequence has recently been reported [Azuma, T., Steiner, L. A. & Eisen, H. N. (1981) Proc. Natl. Acad. Sci. USA 78, 569-573] and is very closely related to the C lambda II amino acid sequence.

Animals↗
PubMed1980

Myeloma with multiple rearranged immunoglobulin kappa genes: only one kappa gene codes for kappa chains.

In many myelomas more than one kappa gene is rearranged (2-5). We are reporting here the results of studies undertaken to determine whether all the rearranged genes are expressed. It was found that in the myeloma NS-1 three different rearranged kappa genes exist. In a subline of NS-1 and several hybridomas produced by fusion of mouse spleen cells with NS-1 it was found that production of NS-1 kappa chains was correlated with the presence of one of the three kappa genes. Loss of this "expressed" gene eliminated the synthesis of the NS-1 kappa chains, loss of one of the other two rearranged kappa genes did not. It is hypothesized, that allelic exclusion (20) of kappa genes generally operates by the functional rearrangement of one kappa gene; other rearrangements are relatively frequent, at least in myelomas, but mostly they are nonfunctional and thus scrambled antibody molecules do not arise.

Animals↗
PubMed1980

Comparison of different rearranged immunoglobulin kappa genes of a myeloma by electronmicroscopy and restriction mapping of cloned DNA: implications for "allelic exclusion".

We have studied the organization and function of different rearranged kappa genes in a myeloma, MOPC-21. Two kappa genes were cloned into Charon 4A and compared with each other and with a cloned germline CK gene by restriction mapping and electron microscopy. One MOPC-21 clone corresponds to the gene coding for the MOPC-21 kappa chain polypeptide; it has the V21 gene joined with the CK gene at the J2 sequence. The other MOPC-21 clone corresponds to a nonfunctional rearranged MOPC-21 kappa gene, except for a lkb deletion, 3' of J4. A similar deletion is also found in a "new" kappa gene present in NS-1, a cellular subclone of MOPC-21. The clone of the "nonfunctional" kappa gene has a V gene which is distinct from V21 which is joined to CK in the vicinity of J2. The undeleted form of this gene codes for a KRNA having the size of mature KmRNA which, however, is not translated into kappa chains. Thus the defect of the "nonfunctional" gene manifests itself at a late step of gene expression. The basis for "allelic exclusion" of antibody genes may simply be the complexity of the processes between genes and gene products, resulting in the expression of only one gene.

Alleles↗