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M L Breitman

Publications and source records attributed to M L Breitman.

At least 73 records · Page 4Linked to original sources
PubMed1987

Gamma-crystallins of the human eye lens: expression analysis of five members of the gene family.

While only two gamma-crystallins have been identified in the human eye lens, molecular studies indicate that the human gamma-crystallins are encoded in a multigene family comprising at least seven closely related members. Sequence analysis of five of these genes has suggested that three (gamma 1-2, G3, and G4) are potentially active, while two (G1 psi and G2 psi) correspond to closely related pseudogenes. Here we report on the detailed structure of a sixth gamma-crystallin gene, G5, and our results obtained with transient expression assays to characterize both the promoter activity and translation products of five members of the gene family. We show that 5'-flanking sequences of G1 psi and G2 psi lacked detectable promoter activity, while the corresponding sequences of G3, G4, and G5 were able to direct high levels of expression of the bacterial chloramphenicol acetyltransferase gene in primary lens epithelia, but not in cultures of nonlens origin. Detailed sequence comparisons indicated that active genes contained several conserved sequence tracts 5' of the TATA box which may constitute functional elements of a lens-specific gamma-crystallin promoter. Expression of the gamma-crystallin coding sequences from the human metallothionein IIA promoter in nonlens cells facilitated characterization of the polypeptides encoded by individual gamma-genes and, in future studies, should permit comparison of these proteins with distinct gamma-crystallins in the human lens.

Amino Acid Sequence↗
PubMed1987

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.

Animals↗
PubMed1987

Metastatic potential of SP1 mouse mammary adenocarcinoma cells is differentially induced by activated and normal forms of c-H-ras.

The mouse mammary adenocarcinoma cell line SP1 was transfected with either the activated T24-H-ras or the normal c-H-ras gene and assessed for metastatic potential in syngeneic CBA/J or nude mice. Unlike the parental control cells, which were tumorigenic but unable to metastasize from a subcutaneous site, all SP1 transfectants expressing the T24-H-ras gene were able to metastasize (predominantly to the lung). In contrast, a much smaller fraction of the clones obtained following transfection with either normal c-H-ras or pSV2neo were metastatic and, significantly, elevated expression of the c-H-ras proto-oncogene did not correlate with acquisition of metastatic potential. We conclude that activated and normal forms of the c-H-ras gene differ in their ability to confer metastatic potential to SP1 mouse mammary adenocarcinoma cells.

Adenocarcinoma↗
PubMed1986

Characterization of the human beta-crystallin gene Hu beta A3/A1 reveals ancestral relationships among the beta gamma-crystallin superfamily.

We report here the detailed structure of a human beta-crystallin gene, designated Hu beta A3/A1, which was isolated and characterized using homologous mouse and bovine beta-crystallin cDNAs. Hu beta A3/A1 consists of six exons, spanning approximately 8 kilobases. The first two exons code for an N-terminal extension of 32 amino acid residues, while the other four encode the four similar structural motifs of the predicted polypeptide. Sequence homologies among the latter four exons and their intron-exon junctions support a model of gene evolution based on two successive exon duplications. Transcription of Hu beta A3/A1 in the eye lens initiates 24 base pairs downstream of a putative TATA box and just 7 nucleotides upstream of a potential initiation codon, generating a single mRNA of approximately 1 kilobase. Comparison of Hu beta A3/A1 with the homologous bovine cDNA and the translation products of the corresponding bovine gene suggests that translation of Hu beta A3/A1 commences at either of two potential initiation codons located in the first and second exons. Differential use of these two codons predicts two polypeptides differing by the presence or absence of 17 amino acid residues at their N-termini.

Amino Acid Sequence↗
PubMed1986

Assignment of the human gamma-crystallin gene cluster (CRYG) to the long arm of chromosome 2, region q33-36.

The gamma-crystallins of the human eye lens are encoded by a multigene family of which at least six genes have recently been assigned to chromosome 2. We have now localized these genes to the distal region of the long arm of chromosome 2 (region q33-36, most probably q34-35) using somatic cell hybrids containing different parts of this chromosome and by in situ hybridization. The gamma-crystallin genes map to the same chromosomal region as IDH-1. Similar linkage exists between the loci Len-1 and Idh-1 on mouse chromosome 1.

Animals↗
PubMed1986

Localization of a beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to the long arm of human chromosome 17.

We have assigned a human beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to chromosome 17 using a panel of 19 human-hamster somatic cell hybrids and blot-hybridization analysis of cell hybrid DNA. Positive probe-hybridization signal was detected in a hybrid that had lost the short arm of human chromosome 17 but retained the long arm, translocated to a hamster chromosome. In addition, in situ hybridization analysis of metaphase chromosome spreads of this cell line suggested that the most probable location for CRYB1 is on the long arm of chromosome 17, in the region q21.

Animals↗
PubMed1985

Replication of pSV2-gpt in COS-1 cells: stability of plasmid DNA in the presence and absence of biochemical selection.

We have previously demonstrated that COS-1 cell lines transformed by pSV2-gpt and maintained under biochemical selection replicate multiple copies of extrachromosomal plasmid DNA (1). We have now examined the replication and stability of this DNA in a representative cell line. In situ hybridization analyses revealed that intense replication of pSV2-gpt occurs in only a small subpopulation of cells and results from bursts of plasmid replication that occur periodically and spontaneously in the cell population. This suggests that COS-1 cells are only semipermissive for pSV2-gpt replication. No correlation was observed between levels of pSV2-gpt replication and the presence or absence of biochemical selection for the Gpt marker. However, growth of cells under nonselective conditions led to a rapid and progressive loss of pSV2-gpt DNA. This loss correlated with segregation of Gpt- revertants that lacked detectable plasmid sequences. Hence, maintenance of pSV2-gpt in the cell line was dependent on continuous biochemical selection. Stable replication of pSV2-gpt could be observed as late as four months after transfection, suggesting that this system might be useful for propagation of cloned DNA in COS-1 cells for extended periods of time. However, by nine months, extensive rearrangements of pSV2-gpt sequences were detected, indicating ultimate instability of the plasmid in the host cells.

Animals↗
PubMed1985

Assignment of human gamma crystallin multigene family to chromosome 2.

The multigene family for human gamma-crystallin has been assigned to chromosome 2 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. Two genomic DNA probes containing human gamma-crystallin gene sequences hybridize to five fragments in human DNA digested with the restriction enzyme EcoRI. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, at least six human gamma-crystallin genes can be mapped to chromosome 2. Data obtained with a hybrid clone containing a mouse-human interspecies translocation suggest that these genes may be clustered together on the long arm of human chromosome 2.

Animals↗
PubMed1985

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.

Amino Acid Sequence↗
PubMed1985

Lens-specific promoter activity of a mouse gamma-crystallin gene.

Crystallins are the major water-soluble proteins in vertebrate eye lenses. These lens-specific proteins are encoded by several gene families, and their expression is differentially regulated during lens cell differentiation. Here we show that a cloned mouse gamma-crystallin promoter is active in lens explants derived from 14-day-old chicken embryos but inactive in a variety of cells of non-lens origin. We also show that sequences required for proper utilization of this promoter are contained between nucleotide positions -392 and +47 relative to the transcription initiation site; deletion of sequences from positions -392 to -171 completely abolishes promoter activity. Since chickens do not have gamma-crystallin genes, the expression of a mouse gamma-crystallin promoter in chicken lens cells suggests that different classes of crystallin genes may be regulated by common lens tissue-specific mechanism(s) independent of species.

Animals↗
PubMed1984

Gamma-crystallin family of the mouse lens: structural and evolutionary relationships.

The heterogeneity inherent among gamma-crystallins of the mouse lens was investigated by sequence analysis of three gamma-crystallin-specific cDNAs. Comparison of the nucleotide sequence of these cDNAs and one previously reported by us revealed that the four gamma-cDNAs share 80-90% homology in nucleotide sequence. The entire 3' half of the coding region shows more variability than the 5' half, whereas the greatest variability is observed in the 3' untranslated region where numerous base substitutions, deletions, and insertions seem to have occurred. Alignment of the amino acid sequences of the four mouse gamma-crystallins according to the known four structural motifs of the major calf gamma-crystallin, gamma-II, suggests that all four mouse polypeptides are structurally very similar to calf gamma-II. However, most of the mouse polypeptides differ from gamma-II by the absence of one amino acid residue, resulting in a shorter connecting peptide between the two globular domains of the protein. Primary sequence alignment also revealed that the four mouse gamma-crystallins are most divergent in the third structural motif of the polypeptide. The significance of these differences in terms of the structure and function of the gamma-crystallins in the mouse lens is discussed.

Amino Acid Sequence↗
PubMed1982

Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells.

We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells. In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.

Animals↗
PubMed1982

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

Animals↗
PubMed1981

PRCII, a representative of a new class of avian sarcoma viruses.

The Poultry Research Center Virus II (PRC II) is a replication-defective avian sarcoma virus with envelope determinants of the A and B subgroups. In nonproducing cells transformed by PRCII the products of the replicative genes gag, pol, and env are not demonstrable, but a single polyprotein of Mr 105,000 (p105) can be detected. P105 contains peptides of the gag proteins p19 and p27 plus transformation-specific sequences. It does not contain peptides of gPr95env of Pr180gag-pol (with the possible exception of one pol peptide). The transformation-specific sequences of p105 are distinct form those of p100 of avian carcinoma virus MH2, of p110 coded for by avian myelocytoma virus MC29, and of p75 or p40 of avian erythroblastosis virus AEV. They also show no resemblance to p60src of Rous sarcoma virus. P105 is phosphorylated on a tyrosine residue and has an associated phosphokinase activity. P105 appears to be capable of autophosphorylation and of phosphorylating homologous immunoglobulin.

Alpharetrovirus↗