Biomedical subjects

J Mucha

Publications and source records attributed to J Mucha.

27 records · Page 2Linked to original sources

PubMed1990

Restriction fragment length polymorphism of chromosome 3 (3p) and the epidermal growth factor receptor gene in human non-small cell lung carcinomas.

We have examined the possible loss of 3p alleles in lung tumor samples from 28 patients with non-small cell lung cancers (non-SCLC), using tumor adjacent lung tissue from the same patients as controls. Of the 14 patients with squamous cell carcinoma only 2 (14%) displayed constitutional heterozygosity at the 3p locus and the tumors of both of these cases did not show reduction to homozygosity. Of the 14 patients with adenocarcinomas, 50% had constitutional heterozygosity, and two of the tumors displayed a loss of heterozygosity. We have also examined restriction fragment length polymorphisms (RFLPs) of the epidermal growth factor (EGF) receptor gene in 29 non-SCLC tumor samples and in the tumor adjacent lung tissue samples obtained from the same cases. Digestion of the DNA samples with the BstEII enzyme and hybridization to a HER-A64-3 probe revealed four different types of polymorphic patterns. We did not, however, detect significant differences in the specific polymorphic bands between tumor and paired non-tumor lung tissues or between the different types of carcinomas.

Blotting, Southern↗

PubMed1990

Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.

Animals↗

PubMed1990

Characterization of three abundant mRNAs from human ovarian granulosa cells.

Three cDNA clones, pHGR122, pHGR11, and pHGR74 containing the coding information for abundant mRNAs were identified from a human ovarian granulosa cell cDNA library. Characterization by nucleotide sequencing revealed that pHGR122 was specific for a collagenase inhibitor and pHGR11 for melanoma-associated antigen ME491. Relative quantification by Northern analysis indicated that collagenase inhibitor mRNA is a major species in granulosa cells. This finding provides evidence for the origin of this protein in follicular fluid as a secretory product of granulosa cells. pHGR11 identified melanoma-associated antigen ME491 as the unexpected product of normal, noncarcinogenic, granulosa cells. pHGR74 has the complete coding information for an unknown protein. Three independent experiments: (i) cell-free translation of pHGR74 RNA; (ii) transcription of suitable restriction fragments followed by cell-free translation; (iii) hydrolysis of the cell-free translation product of pHGR74 RNA by endoproteinase Lys-C, identified one open reading frame coding for an acidic, highly hydrophilic protein of 111 amino acid residues. pHGR74 mRNA is expressed in human testis, prostate, seminal vesicle, and ovarian granulosa cells. A comparative Southern analysis indicates pHGR74 mRNA is species specific and encoded by a single-copy gene.

Amino Acid Sequence↗

PubMed1989

Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method.

We have developed a very rapid procedure for DNA sequence analysis of induced mutations in a typically large mammalian gene. We are able to determine the nature of chemical carcinogen-induced point mutations in the 25 kb Chinese hamster ovary (CHO) cell dihydrofolate reductase gene within two days starting with 5 to 10 x 10(6) cells. The approach is based on the use of rapidly prepared total RNA from which a 730 bp dhfr cDNA is synthesized by reverse transcriptase and amplified by the polymerase chain reaction (PCR) procedure. Genomic DNA can simultaneously be prepared from the same cells. The amplified double-stranded cDNA is then sequenced directly by the dideoxy method using Taq polymerase in the Thermal Cycler (Perkin Elmer/Cetus). We have previously shown that nonsense codons in the dhfr coding sequence often result in greatly reduced steady-state levels of dhfr mRNA (2). The methods described are suitable for mutants of this type which contain only 1 to 2 copies of mRNA per cell. This approach is readily adaptable to other selectable genes and other cell types provided the necessary primers can be prepared.

Animals↗

PubMed1988

Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2.

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.

Amino Acid Sequence↗

PubMed1986