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Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 235 records · Page 13Linked to original sources
PubMed1980

A mouse globin gene promoter is functional in SV40.

We have constructed two SV40 recombinants carrying a complete mouse alpha-globin gene with its presumptive promoter region. In one recombinant the globin gene can be transcribed either from its own promoter or from the adjacent viral late region promoter. In the other efficient globin expression should depend only upon the promoter carried by the chromosomal globin gene. We show that both viruses direct the synthesis of functional globin mRNA in infected monkey kidney cells and that this mRNA has a 5' terminus indistinguishable from that of authentic globin mRNA. These results suggest that the cloned globin gene contains a functional promoter that is accurately recognized in monkey kidney cells.

Animals↗
PubMed1980

Variation in the crossover point of kappa immunoglobulin gene V-J recombination: evidence from a cryptic gene.

Analysis of amino acid and nucleotide sequences of kappa immunoglobulin chains and their genes has led to the hypothesis that the exact site of V-J joining in these genes varies and that this variation is partially responsible for generating amino acid diversity at the recombination site. To assess this hypothesis we have cloned and determined the sequence of one of the two V-J recombinant genes carried by the plasmacytoma MOPC173 and the V region germline precursor of this gene. We find that this V region has been joined to a J segment at a crossover point that indeed differs from the one previously described. This recombination has occurred in such a way as to produce an out of phase, missense reading frame and, hence, a cryptic light chain gene. This result directly supports the cross-over-point variation hypothesis but also indicates that the flexibility of this reaction is accompanied by a cost to the organism in terms of the generation of missense genes.

Amino Acid Sequence↗
PubMed1980

Immunoglobulin gamma 1 heavy chain gene: structural gene sequences cloned in a bacterial plasmid.

Immunoglobulin gamma 1 heavy chain cDNA was cloned into an Escherichia coli plasmid pCR1 and its nucleotide sequence was determined. The hybrid plasmid contained approx. 900-bases-long gamma 1 chain cDNA sequence, including the complete sequence of the CH2 and CH3 domains and the 3' untranslated region, and partial sequence of the CH1 domain. The nucleotide sequence predicts an extra lysine at the carboxyl-terminus of the gamma 1 chain. Comparison of the nucleotide sequence of 3' untranslated regions of the immunoglobulin gamma 1 chain and kappa light chain showed a significant homology although lengths are quite divergent.

Animals↗
PubMed1980

Immunoglobulin V/J recombination is accompanied by deletion of joining site and variable region segments.

A site-specific recombination event is responsible for the somatic activation of immunoglobulin genes and for generating a major share of immunoglobulin gene diversity. Although several possible mechanisms can be proposed to account for this process, recombinatio accompanied by deletion is a particularly attractive mechanism because it might utilize inverted repeat sequences noted on the 3' side of all variable regions and on the 5' side of all joining site segments thus far studied. Testing this model is complicated by the fact that antibody cells are at least diploid and gene segments on the inactive chromosome can obscure deletions occurring within the active gene. Accordingly, we have screened several immunoglobulin-producing plasmacytoma lines to select those in which both chromosomes are rearranged. By using appropriate cell lines and variable and joining region probes in in situ hybridization experiments, we show that recombination is accompanied by the deletion of both variable and joining region genes. These experiments also allow us to map the site of V/J recombination of several active immunoglobulin genes and suggest an order and orientation for variable, joining, and constant region sequences.

Animals↗
PubMed1980

Evolutionary approach to the question of immunoglobulin heavy chain switching: evidence from cloned human and mouse genes.

We have used cloned mouse and human heavy chain genes to identify regions of conserved homology between mouse and man and between mu and alpha genes. Using heteroduplex mapping, we find that coding segments for homologous domains appear to have been well conserved between the two species, but the short intervening sequences that separate domains have diverged considerably. These studies also identify extensive regions of homology 5' to both mu and alpha constant region genes that are conserved between the two genes as well as between mouse and man. These segments encompass regions in which mu/alpha recombination occurs during the heavy chain class switch, and their extensive homology may be relevant to this process.

Animals↗
PubMed1980

In vitro transcription of normal, mutant, and truncated mouse alpha-globin genes.

We have transcribed various alpha-globin gene-containing templates by using a simplified in vitro system and have shown that the 5' end of each of the RNA polymerase II-specific transcripts corresponds to that of authentic alpha-globin mRNA. Using a truncated alpha-globin gene fragment, we then localized the promotor region of the adult alpha-globin gene to a 138-base-pair fragment of alpha-globin DNA, 141 nucleotides of which precede the initiation site. In contrast, when two naturally occurring alpha-globin gene mutants were tested as templates, no RNA polymerase II-dependent initiation was detected. Comparison of the nucleotide sequences of the normal and mutant alpha-globin genes has allowed us to focus upon nucleotide positions that might be essential for promoter activity.

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↗
PubMed1980

Unusual alpha-globin-like gene that has cleanly lost both globin intervening sequences.

We have cloned and determined the nucleotide sequence of a mouse alpha-globin-like gene that entirely lacks the two intervening sequences that interrupt all globin genes thus far examined. The fact that this gene, alpha-3, is closely homologous to the normal adult alpha gene sequence suggests that it arose after the alpha/beta divergence and that it therefore must have lost its intervening sequences. The further fact that these intervening sequences have been lost cleanly--that is, in according with the G-T/A-G splicing rule of RNA--suggests, among other possibilities, that their loss may have been brought about by a gene conversion event involving the mediation of mature globin mRNA or its cDNA cognate. We propose such a mechanism that would permit the loss of either or both intervening sequences independently. Only the loss of both, however, should result in the inactivation of the globin gene, as seems to be the case with alpha-3.

Animals↗
PubMed1979

A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation.

The active gene for a kappa light chain is formed by a somatic recombination event that joins one of several hundred variable region genes to one of a series of recombination sites (J-segments) encoded close to the kappa constant region gene. The nucleotide sequences of cloned germ line and somatically recombined genes define the precise organisation of these genetic segments and the site and nature of the recombination event that joined them. Apart from somatic recombination, no further alteration of ther germ line sequence has occurred. The J-segment is of special interest as it encodes signals for both DNA and RNA splicing and provides a means of generating further immunoglobulin gene diversity.

Animals↗
PubMed1979

The evolution and sequence comparison of two recently diverged mouse chromosomal beta--globin genes.

We have determined the entire nucleotide sequence of a cloned mouse beta--globinminor gene and compared it to the closely related sequence of the betamajor gene. These two genes differ by nine amino acids and presumably evolved from a common ancestral gene as recently as 50 million years ago. Since these genes are closely linked and coordinately expressed, they provide an especially favorable opportunity to assess selection and mutation as these processes affect genes under similar constraints. We find that evolution has preserved these two genes in two short segments of DNA which include their immediately adjacent flanking regions. These regions presumably encode functions that are necessary for proper globin gene expression. In contrast, the more distal flanking sequences and major segments of the long intervening sequences have diverged much more sharply. The homology pattern in these genes also provides considerable insight into the mechanisms by which less constrained nucleotide sequences diverge rapidly. Change in such regions apparently occurs less by point mutation than by insertion, deletion and duplication of relatively short segments of the genome.

Amino Acid Sequence↗
PubMed1979

The complete sequence of a chromosomal mouse alpha--globin gene reveals elements conserved throughout vertebrate evolution.

The mammalian alpha- and beta--globin genes are thought to have evolved from a common ancestral sequence by a duplication event that occurred over 500 million years ago. We have now determined the entire nucleotide sequence of a cloned mouse alpha--globin gene, including regions that flank and interrupt the coding sequence, and have compared this sequence with the sequences of the two mouse beta--globin genes (Konkel, Tilghman and Leder, 1978; Konkel, Maizel and Leder, 1979). Like the two beta genes, the alpha gene is interrupted by two intervening sequences at precisely homologous positions, suggesting that these interruptions were present and have been preserved throughout vertebrate evolution. While the alpha and beta genes conserve considerable (approximately 55%) sequence homology in their coding regions, this homology--with certain interesting exceptions--is lost in the highly divergent flanking and intervening sequences. These exceptions are short preserved sequences positioned in such a way that they might encode signals for transcriptional initiation, poly(A) addition and RNA splicing. Furthermore, a comparison of the recently divererged beta genes and the long separate alpha gene allows us to distinguish two clearly different modes of nucleotide sequence change in evolution: a fast mode which is characterized by drastic sequence alterations involving deletions and insertions, and a slow mode which preserves sequence homology to a large extent and involves mainly point mutations.

Animals↗
PubMed1979

Splicing and the formation of stable RNA.

To determine whether RNA splicing plays an obligatory role in gene expression, we have constructed a series of SV40-transducing viruses carrying various combinations of splice junctions derived from the viral genome and a mouse globin gene. All of the viruses that retain at least one functional splice junction, derived from either the viral or the mouse genome, encode stable hybrid RNAs. In contrast, a virus from which all the splice junctions have been removed fails to produce any detectable stable RNA. These results suggest that splicing is a prerequisite for stable RNA formation.

Animals↗
PubMed1979

SV40 recombinants carrying rabbit beta-globin gene coding sequences.

We have constructed and propagated two SV40 recombinants in which different portions of the viral late gene region are replaced by a rabbit beta-globin coding sequence derived from a cloned cDNA (Maniatis et al., 1976). One of these recombinants, which retains the promoter, leader and intervening sequences for a viral late mRNA, directs the synthesis of a stable SV40-globin hybrid transcript. Using a sensitive radioimmunoassay, we have shown that this globin RNA is translated in infected monkey cells. A second recombinant, which retains the late region promoter but lacks the RNA splicing region, produces neither a stable globin transcript nor any detectable beta-globin. These experiments indicate that some sequence within a 500 bp SV40 segment, presumably the splicing region, is required for the accumulation of stable mRNA. They also demonstrate how hybrid transducing viruses can be used both to characterize viral regulatory sequences and to produce new gene products in cultured cells.

Animals↗
PubMed1979

SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene.

We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal.

Animals↗
PubMed1979

Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene.

Immunoglobulin kappa chain gene formation involves site-specific somatic recombination between one of several hundred germ-line variable region genes and a joining site (or "J segment") encoded close to the constant region gene. We have cloned and determined the nucleotide sequence of major portions of the recombination region of the mouse kappa gene and discovered a series of five such J segments spread out along a segment of DNA 2.4 kilobases from the kappa constant region gene. These J segments encode the 13 COOH-terminal amino acids of the variable region, probably including amino acids involved in the antigen combining site and in heavy/light chain contacts. The J segments also display striking sequence homology to one another in both their coding and immediately flanking sequences. Major elements of a short palindrome--CAC(TA)GTG--are preserved adjacent to the recombination sites of both variable and J region genes and constitute inverted repeats at both ends of the sequences to be joined. These palindromes can be written as a hypothetical stem structure that draws variable and J regions together, providing a possible molecular basis for the DNA joining event. Four of the J segments that we have discovered encode amino acid sequences already found in myeloma proteins. By altering the frame of recombination, we can account for additional light chain amino acid sequences, suggesting that the V/J joining event might generate antibody diversity somatically both by using different combinations of variable and J region genes and by using alternative joining frames.

Animals↗