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Biomedical subjects

D H Hamer

Publications and source records attributed to D H Hamer.

At least 73 records · Page 4Linked to original sources
PubMed1984

SV40-alpha-globulin hybrid minichromosomes. Differences in DNase I hypersensitivity of promoter and enhancer sequences.

The structure of minichromosomes from an SV40 recombinant carrying a functional mouse alpha-globulin gene in its late coding region was studied by DNase I treatment of isolated nuclei from infected cells. Ten prominent DNase-I-hypersensitive sites were detected in the inserted globin fragment, six in the region upstream from the transcription initiation site and four within the gene itself. Corresponding sites were not seen in naked DNA, nor in minichromosomes without this insertion. However this hypersensitivity pattern differs from that observed for mouse alpha-globin in Friend erythroleukemia cells before or after induction. The recombinant minichromosome also displayed eight hypersensitive sites, indistinguishable from those seen in unsubstituted SV40, in the viral sequences containing the origin of replication and the regulatory signals for early and late viral transcription. The cleavage sites in SV40 occur predominently in the origin-promotor-enhancer region, whereas those in the globin fragment were more dispersed and less pronounced. This suggests that the specific local opening of this region in the viral chromatin is not due solely to the presence of active promoters, but is associated with the function of the enhancer element.

Alpha-Globulins↗
PubMed1984

Chromosomal location of human metallothionein genes: implications for Menkes' disease.

Human metallothioneins are encoded by a complex multigene family. The chromosomal location of these genes has been determined by gel transfer hybridization analysis of the DNA from human-rodent cell hybrids. Chromosome 16 contains a cluster of metallothionein sequences, including two functional metallothionein I genes and a functional metallothionein II gene. The remaining sequences, including a processed pseudogene, are dispersed to at least four other autosomes. The absence of metallothionein sequences from the X chromosome indicates that Menkes' disease, an X-linked disorder of copper metabolism, affects metallothionein expression by a trans-acting mechanism.

Animals↗
PubMed1984

Duplicated heavy metal control sequences of the mouse metallothionein-I gene.

We present evidence that two distinct regions of the DNA upstream from the mouse metallothionein-I gene contain metal-responsive regulatory sites. This result was obtained by analyzing a systematic series of deletion, insertion, duplication, and clustered point mutations introduced into cultured cells on a simian virus 40 plasmid vector. The two upstream regions contain a duplicated evolutionarily conserved DNA sequence. While either upstream region is sufficient to confer heavy metal responsiveness, both are required to give maximal levels of induced transcription.

Amino Acid Sequence↗
PubMed1983

Cloning and sequence analysis of two monkey metallothionein cDNAs.

We have isolated two metallothionein (MT) cDNA clones copied from the RNA of cadmium-resistant monkey kidney cells. The complete DNA sequences of these clones show that they encode two distinct MTs. One clone appears to represent monkey MT-II, as shown by its close homology to the human MT-II sequence, whereas the second may correspond to monkey MT-I or a related variant metallothionein. Conserved sequences were identified in both the 5' and 3' untranslated regions of these clones.

Animals↗
PubMed1983

Regulation of a metallothionein-growth hormone hybrid gene in bovine papilloma virus.

We have constructed bovine papilloma virus recombinants carrying a hybrid gene in which human growth hormone structural sequences are fused to the promoter and presumptive control region of the mouse metallothionein-I gene. Mouse cells transformed with the recombinants synthesize metallothionein-growth hormone hybrid mRNA with the same 5' end as metallothionein mRNA. Hybrid mRNA is inducible by cadmium but not by dexamethasone, whereas the chromosomal metallothionein genes in the same cells are inducible by both agents. This indicates that heavy metals and glucocorticoids regulate the mouse metallothionein-I gene by independent mechanisms. Furthermore, because the transformed cells produce large quantities of human growth hormone polypeptide, it appears that bovine papilloma virus-metallothionein vectors may be useful for obtaining the efficient and regulatable expression of other eukaryotic gene products. We have constructed recombinants that allow the insertion of new structural sequences under the control of the metallothionein promoter.

Animals↗
PubMed1982

Construction and expression in vivo of an internally deleted mouse alpha-fetoprotein gene: presence of a transcribed Alu-like repeat within the first intervening sequence.

An alpha-fetoprotein (AFP) 'minigene' was constructed by joining the first two exons along with 0.9 kilobases of 5' flanking sequence of the mouse AFP gene to its last exon and 0.4 kilobases of 3' flanking sequence. This 'minigene' was tested for activity by inserting it into an SV40 vector and infecting African green monkey kidney cells. Correct initiation, termination, polyadenylation and splicing were observed. Furthermore a 220 nucleotide transcript was detected and mapped to a mouse Alu-like or B1 repeat on the opposite strand to that encoding the AFP gene in the first intervening sequence.

Animals↗
PubMed1982

Abnormal RNA splicing causes one form of alpha thalassemia.

The alpha 2-globin gene from a patient with alpha thalassemia contains a pentanucleotide deletion in intron 1 immediately adjacent to exon 1. We tested the functional consequences of this mutation by introducing the thalassemic gene, along with its normal counterpart as a control, into cultured monkey cells on SV40 plasmid vectors. Both genes are expressed, at similar levels, into globin RNA with the correct 5' and 3' ends. However, while most of the normal transcripts are appropriately processed, the thalassemic transcripts are abnormally spliced from a 5' donor site in the middle of exon 1 to the normal 3' acceptor site. This results in the synthesis of a truncated RNA incapable of encoding a normal globin polypeptide. The alternative donor, also used at a low level in monkey cells transfected with the normal gene, shows strong homology to the consensus donor sequence characteristic of many eucaryotic splice junctions. No unspliced or partially spliced thalassemic RNA was detected, indicating that recognition of this site is efficient and does not block removal of intron 2. The alternatively spliced RNA was also found in bone marrow RNA from the alpha-thalassemic patient, although not in that from a normal individual or a beta +-thalassemic patient. The thalassemic phenotype of the patient therefore results from abnormal RNA splicing owing to the deletion of the first splice donor signal.

Base Sequence↗
PubMed1982

Properties of human growth hormone polypeptides: purified from pituitary extracts and synthesized in monkey kidney cells and bacteria.

Several forms of human GH (hGH) have been elucidated by extraction of human pituitary and recombinant DNA techniques. In the present study we have characterized five of these hGH polypeptides by gel filtration, RIA, and radioreceptor assays. These include extractable pituitary hGH and its naturally occurring 20K variant. Two hGH polypeptides were produced from naturally occurring human genes in simian kidney cells (SV-hGH 1 and 2) and another preparation was produced from a partially synthesized gene in bacteria (E. coli-hGH). As predicted from their known DNA sequences, naturally occurring pituitary hGH, SV-hGH 1, and E. coli-hGH migrated as a single peak on Sephadex G-100 column and had the same immunological and receptor-binding properties. By contrast, SV-hGH 2 (14 dispersed amino acid substitutions) and the 20K variant (amino acid residues 32-46 deleted from hGH) contained more higher molecular weight components and had diminished immunological and receptor-binding potency. SV-hGH 2 differed from the 20K variant by having even lower immunological potency and containing more of the higher molecular weight component. These variant forms of hGH may provide an explanation for the heterogeneity of both pituitary and plasma hGH.

Animals↗
PubMed1982

Regulation in vivo of a cloned mammalian gene: cadmium induces the transcription of a mouse metallothionein gene in SV40 vectors.

Cadmium and other heavy metals induce metallothionein synthesis in mammalian cells. We have cloned a mouse metallothionein-I gene from a cadmium-resistant line of Ltk- cells and inserted it into viral and plasmid SV40 vectors. These recombinant molecules were introduced into cultured monkey cells and the expression of the foreign metallothionein gene was analyzed, at the RNA level, in the presence and absence of cadmium. The results show that the cloned gene retains its cadmium inducibility and that the regulation of this gene occurs primarily at the transcriptional level.

Animals↗
PubMed1981

Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants.

We have constructed simian virus 40 recombinants carrying two different human growth hormone (hGH) genes. Monkey kidney cells infected with these recombinants synthesize, process, and secrete hGH. The product of gene 1, which has coding sequences identical to those of a cloned hGH complementary DNA, is indistinguishable from pituitary hGH by several criteria. The product of gene 2, which is predicted to encode a variant protein, is less immunoreactive than pituitary hGH but binds efficiently to hGH cell surface receptors. These results show that gene 2 has the potential to be expressed into a previously unidentified form of hGH. They also demonstrate that it is possible to produce a mature hormone by gene transfer in eukaryotic cells and indicate the utility of the simian virus 40-monkey cell system for producing and characterizing secreted animal cell proteins.

Animals↗
PubMed1981

Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector.

We have constructed a simian virus 40 recombinant carrying a fragment of DNA from hepatitis B virus. Cultured monkey kidney cells infected with this recombinant produce hepatitis B surface antigen. The antigen is excreted into the culture medium as 22-nm particles with the same physical properties, antigenic composition, and constituent polypeptides as those found in the sera of patients with type B hepatitis.

Animals↗
PubMed1981

The organization and evolution of cloned globin genes.

The globin genes represent a complex set of sequences that are expressed in a coordinate fashion during the development of red blood cells. while this complex family of genes may consist of as many as ten to fourteen members [34], three of these genes have now been cloned and their entire nucleotide sequence determined. As was initially observed in the case of beta globin major gene, all are encoded in three distinct coding blocks separated by two intervening sequences of DNA. Their intervening sequences of DNA are preserved, with respect to location, but are widely divergent, with respect to size and sequence. The divided information in each gene is edited and spliced together at the level of its initial RNA transcript which is complementary to the entire gene sequence including its intervening sequences. Structural correlation analyses have allowed us to identify sites in all three genes that might be responsible for the initiation of transcription, RNA splicing, and poly A addition. The function of these sites has been tested by cloning these genes in an animal virus vector SV40. Such animal virus hybrids have been used to infect tissue culture cells and have directed the synthesis of both alpha and beta mouse globin in cells of monkey origin. These studies indicate that such signals operate across species barriers and further indicate that the animal virus vector system will be useful in elucidating their function.

Amino Acid Sequence↗
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

Eukaryotic gene transfer and the new genetics.

The function of specific eukaryotic DNA sequences can be assessed by the following procedure. First, the gene of interest is purified, usually by cloning in bacteria, and its structure is determined. Second, biochemical techniques are used to introduce mutations into the regions of interest. Finally, the normal and mutant genes are introduced into an appropriate eukaryotic cell where their expression can be examined in detail. In this paper I review some of the techniques that can be used for gene transfer, and speculate on how they might be applied to parasites.

Cell Fusion↗