[Sex determination of human blood micro-stains and single hairs using specific DNA amplification with polymerase chain reaction].

Amplification of Y chromosome specific DNA in vitro enables a rapid and reliable sex determination of human minute traces such as blood stains and hairs. In presence of male DNA a band of 154 bp is visualized by agarose gel electrophoresis after amplification, this band is lacking in case of female DNA alone. Amplification of a sex independent DNA locus (such as a fragment from the alcohol dehydrogenase gene) generates identical reaction products for both sexes.

Genotyping of human class I alcohol dehydrogenase. Analysis of enzymatically amplified DNA with allele-specific oligonucleotides.

Large inter-individual differences are noted in the susceptibility to alcohol-related problems. Part of this variation may be due to the different isoenzyme patterns of the alcohol-metabolizing enzymes and, consequently, different pharmacokinetics of alcohol degradation. We have used the polymerase chain reaction and oligonucleotide hybridization to amplify and analyze class I alcohol dehydrogenase isoenzyme-specific genomic DNA.

The v-mos and H-ras oncogene expression represses glucocorticoid hormone-dependent transcription from the mouse mammary tumor virus LTR.

We have subjected the viral mos oncogene (v-mos), the activated human H-ras oncogene [H-ras (A)] and the normal human H-ras protooncogene [H-ras (N)] to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells. Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium. The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation.

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype.

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells.

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo.

Persistence, methylation and expression of vitellogenin gene derivatives after injection into fertilized eggs of Xenopus laevis.

We report the fate of different derivatives of the vitellogenin genes after injection into fertilized eggs of Xenopus. We injected a constructed minigene as well as a 5' fragment of the A2 vitellogenin gene. The minigene survives in embryogenesis much better than the 5' A2 fragment and is retained more frequently and at a higher level in frog tissues.

Scattering of repetitive DNA sequences in the albumin and vitellogenin gene loci of Xenopus laevis.

We have analyzed middle repetitive DNA in the albumin and vitellogenin gene families of Xenopus laevis. Mapping specific repetitive DNA sequences derived from introns of the A1 vitellogenin gene reveals that these sequences are scattered within and around the four vitellogenin genes (A1, A2, B1 and B2) and the two albumin genes (74 kd and 68 kd). Three repetitive DNA elements present in the A1 vitellogenin transcriptional unit are also located in introns of the 74 kd albumin gene.

Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%.

Identification, organization and processing intermediates of the putative precursors of Xenopus vitellogenin messenger RNA.

To understand the mechanism of estrogen-induced activation of the vitellogenin genes in the liver of Xenopus, it is essential to characterize the transcriptional products of these genes. In this paper we describe large nuclear RNAs containing vitellogenin mRNA sequences as revealed by hybridization of cloned vitellogenin cDNAs to nuclear RNA separated on agarose gels. Putative vitellogenin mRNA precursors, which are recovered as poly(A)-containing RNA, have been identified for the four known vitellogenin mRNAs.

Intergrated stereological and biochemical studies of hepatocytic membranes. I. Membrane recoveries in subcellular fractions.

Previous attempts to relate the structure and function of hepatocytic membranes have compared biochemical data of fractions to morphological data derived from either intact tissue or fractions. The effects of the original homogenization aside, biochemical recoveries comparing membrane marker enzymes of the homogenate to subsequent fractions suggest a general conservation of activity. A sterological study was undertaken to estimate membrane surface areas in the intact tissue, homogenate, and fractions of the same livers and then to test the comparability of these data with membrane marker enzymes by calculating both morphological and biochemical recoveries.