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%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase.

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. The sterological data were corrected for errors due to section thickness and compression. The average total membrane sufrace area per 1 g of liver was 9.3 m2 in the intact tissue (T), 7.8 m2 in the homogenate (H), and 7.4 m2 in the fractions (F); recoveries for the membrane surface areas thus averaged 96% for the (F/H) and 81% for the (F/T) comparisons. In homogenate and fractions, the differentiability of membranes by morphological criteria was limited to rough- and smooth- surfaced membranes, as well as outer and inner mitochondrial membranes. The recoveries of rough-surfaced membranes were 101% for F/H and 92% for F/T; those of smooth-surface membranes were 89% for F/H and 107% for F/T. For mitochondrial membranes, a recovery of 100% for F/H was obtained, whereas it amounted to only 54% for F/T. With respect to F/H, the membrane recoveries compare well with the marker enzyme recoveries obtained biochemically. The extension of recovery calculations to the intact tissue (F/T) revealed satisfactory conservation of the procedures of homogenization and fractionation; it indicates, however, that a shift of a substantial part of mitochondrial membranes to the pool of unidentifiable smooth membranes may occur on homogenization.

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. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

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. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.

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 method unambiguously distinguishes between different allelic variants and thus provides a new means of elucidating the alcohol dehydrogenase isoenzyme pattern of humans.

[Sex determination of human blood micro-stains and single hairs using specific DNA amplification with polymerase chain reaction]. / Geschlechtsbestimmung an menschlichen Mikroblutspuren und Einzelhaaren mit Hilfe gezielter DNA Amplifikation durch die 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. This shows that the absence of a band is not due to the lack of trace DNA. It is possible to perform this technique with as little as 0.5 microliters of blood or with a single hair.

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. Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone. Measurements of the transcriptional rates in nuclei from LTR v-mos and LTR H-ras (A) transfected cells showed a repression of LTR v-mos and LTR H-ras (A) transcription after the initial stimulation by hormone. LTR H-ras (N) transcription was not affected. An independently transfected LTR H-2Ld construct in LTR v-mos or LTR H-ras (A) containing cells is also transcriptionally repressed. These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone-dependent MMTV LTR transcription.

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. The mosaic distribution of the foreign DNA in different frog tissues indicates that no integration occurred before the first cleavage stage. The persisting DNA may be partially integrated but is mostly found in an episome-like form. This unintegrated form is not supercoiled and is rearranged. Methylation of the Hpa II sites prior to injection has no influence on the survival of the injected sequences and the Hpa II sites of the surviving DNA are unmethylated irrespective whether the injected DNA was methylated or not. Whereas the derivatives are transcribed in embryos, they cannot be activated by estrogen in the liver of young frogs.

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. From electron microscopic analysis of R loops, prepared between enriched mRNA precursors and cDNA specific for the A1 vitellogenin mRNA, we conclude that the precursor molecules contain sequences complementary to vitellogenin mRNA which are interrupted by additional RNA segments probably representing transcribed introns. Within the 3.7 kb of the 3' end of the A1 vitellogenin mRNA we have discovered seven large and at least five small transcribed introns. Some of the R loops have been found to contain only a few transcribed introns, and we assume that they represent processing intermediates. Comparison of these putative intermediates suggests that the splicing order of different introns does not follow a single pathway.

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. This apparently random distribution of middle repetitive DNA in the two gene families suggests that the analyzed sequences are not involved in gene regulation, but rather that they might represent unstable genetic elements. This hypothesis is further supported by the finding that size polymorphism in the A1 vitellogenin gene and in the 74 kd albumin gene is correlated with the presence or absence of repetitive DNA.