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K Bonham

Publications and source records attributed to K Bonham.

13 recordsLinked to original sources
PubMed1993

Organization and analysis of the promoter region and 5' non-coding exons of the human c-src proto-oncogene.

In order to help clarify the cellular mechanisms that regulate expression of the human c-src proto-oncogene, we have isolated a series of overlapping genomic clones that contain the c-src promoter region, as well as three previously uncharacterized exons. These exons encode the 350-bp 5' untranslated region of the c-src mRNA and span 35 kb of genomic DNA, extending the human c-src locus to approximately 60 kb. Subcloning and sequence analysis of the 5' flanking region of the gene revealed a high GC content and several consensus Sp1 and AP2 binding sites. However, TATA or CAAT boxes were not present, a characteristic shared by other GC-rich promoters. Promoter-CAT constructs demonstrated that the promoter was functional in transfection assays and that its activity was dependent on correct orientation. CAT-promoter deletion constructs were used to define the 5' boundary for maximal promoter activity and to reveal the presence of both positive and negative regulatory elements. S1 analyses of human c-src mRNA from cell lines indicated that multiple transcription start sites were utilized.

Base Sequence↗
PubMed1992

Overexpression of three ubiquitin genes in mouse epidermal tumors is associated with enhanced cellular proliferation and stress.

A mouse ubiquitin clone that recognizes multiple transcripts overexpressed in murine tumors compared to normal epidermis was isolated by differential screening of complementary DNA libraries from mouse squamous cell carcinomas. Coding region probes detected five ubiquitin transcripts. Oligonucleotides were designed for unique parts of three mouse ubiquitin gene complementary DNA clones. The overexpressed transcripts at 2.4, 2.8, 6.4, and 7.8 kilobases (kb) were detected by an oligonucleotide specific for a mouse UbC polyubiquitin clone. A 1.2-kb UbB overexpressed transcript was detected by an oligonucleotide for a mouse four-unit polyubiquitin, and a 0.7-kb UbA overexpressed transcript was recognized by an oligonucleotide for the mouse ubiquitin carboxyl-extension protein of 52 amino acids. All three classes of transcripts were induced in mouse skin by the hyperproliferative agent ethylphenyl propionate and by the tumor promoting agent 12-O-tetradecanoylphorbol-13- acetate. Heat shock of cultured keratinocytes induced both the 6.4- and 7.8-kb transcripts recognized by the UbC-specific oligonucleotide. Consistent with the overexpression of the ubiquitin transcripts, the level of free ubiquitin protein, as determined by Western analysis, was elevated in the tumors and proliferating epidermis as compared to normal epidermis. Our results indicate that the overexpression of ubiquitin genes could be related to a sustained state of proliferation and stress in the tumors compared to the normal resting epidermis.

Alkynes↗
PubMed1989

Activation of the cellular Harvey ras gene in mouse skin tumors initiated with urethane.

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.

Animals↗
PubMed1988

Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region.

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.

Amino Acid Sequence↗
PubMed1987

Metallothionein gene expression in fish cell lines: its activation in embryonic cells by 5-azacytidine.

We have investigated the regulation of metallothionein gene expression in two fish cell lines. Rainbow trout hepatoma (RTH) cells synthesized metallothionein in response to heavy metal exposure. The maximum level of metallothionein synthesis detected during zinc exposure was much greater than during cadmium exposure. The time-courses of metallothionein synthesis were different for the different metal inducers, suggesting that metallothionein may be differentially regulated by cadmium and zinc in these cells. The metal-induced synthesis of metallothionein was correlated with increased translational activity and accumulation of metallothionein-mRNA, suggesting that metallothionein may be regulated at the transcriptional and post-transcriptional levels in RTH cells. Chinook salmon embryo (CHSE) cells, unlike RTH cells, did not synthesize metallothionein or metallothionein-mRNA in response to heavy metal exposure. However, when these cells were treated with 5-azacytidine prior to heavy metal exposure, the synthesis of metallothionein was induced, suggesting that DNA methylation may play a role in metallothionein gene expression in fish.

Animals↗
PubMed1987

The rainbow trout metallothioneins: molecular cloning and characterization of two distinct cDNA sequences.

The rainbow trout hepatoma (RTH) cell line responds to heavy metals such as zinc and cadmium by synthesizing the ubiquitous thiol-rich protein metallothionein (MT). From this cell line we have isolated two full-length cDNA clones, tMT-A and tMT-B, which encode two similar but distinct trout MTs. The clones were isolated by cross-homologies between the trout MT mRNAs and a human MT riboprobe. Clones tMT-A and tMT-B code for proteins of 61 and 60 amino acids, respectively; the one extra amino acid in tMT-A is due to an apparent insertion at position 31 of the protein. There are also two other amino acid changes between the two isoforms. Overall, the coding regions show extensive homologies to mammalian MTs, especially at the cysteine residues and at a core sequence at the boundary of the two domains. However, closer examination reveals a number of significant differences in positions usually invariant in the mammalian MTs. Northern blot analysis of RNA from metal-treated RTH cells demonstrated MT-mRNA is induced to high levels by zinc, low levels by cadmium, and minimally by copper. In contrast, intraperitoneal injections of rainbow trout demonstrated that all three metals induce MT-mRNA to comparable levels in the liver. Southern blot analysis of trout DNA cleaved with three restriction enzymes suggests that the trout family of MT genes is probably limited to these two members.

Amino Acid Sequence↗
PubMed1986

Heavy metal-induced gene expression in fish and fish cell lines.

Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl2 and ZnCl2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW = 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 109Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium. We suggest that both MT and MIP may function in detoxification of heavy metals.

Amino Acids↗
PubMed1984

Induction of metallothionein and metallothionein mRNA in rainbow-trout liver following cadmium treatment.

A low-molecular-weight cadmium-binding protein was induced in the livers of rainbow trout (Salmo gairdnerii) following a series of intraperitoneal injections of cadmium chloride. The subsequent purification and amino acid analysis of this protein showed it to be a true metallothionein. As in higher organisms, two major forms of metallothionein appeared to be present in the liver following cadmium treatment. Following a similar induction procedure total RNA was also isolated and shown to contain high levels of metallothionein-mRNA activity when assayed in a wheat-germ cell-free translation system. This activity was present in the Poly-A+-containing fraction of the total RNA. The bulk of this mRNA activity was shown to be in the 8-10S region of a sucrose gradient.

Animals↗