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After fertilization the cone shaped sperm nucleus is transformed into a spheroidal male pronucleus which fuses with the female pronucleus reestablishing the diploid genome. These morphological changes are correlated with biochemical transitions of sperm-specific chromosomal proteins. To obtain information on the rearrangements of chromosomal proteins after fertilization, we have followed sperm-specific nonhistones (Sp NHCP) and the sperm-specific histones (SpH) in zygotes harvested at different times post-insemination (p.i.). The acquisition of proteins synthesized de novo was determined during the time of male pronucleus formation, estimated to occur until 30 min p.i. The results obtained may be summarized as follows: 1. The fate of Sp NHCP, followed by Western blot analysis with polyclonal antibodies obtained in rabbits against the whole complement of Sp NHCP, indicates that the majority of Sp NHCP are lost shortly p.i., except two proteins of 58 Kd and 61 Kd that were not distinguishable from eggs NHCP because a crossreaction was obtained. Acidic proteins synthesized de novo were bound to chromatin between 3 and 30 min p.i., and the majority of these proteins comigrated with egg NHCP in SDS/PAGE. 2. The histones from sperm differ from their counterparts found in unfertilized eggs, both in their amino acid composition and their microheterogeneity in bidimensional gels. Sperm contain the five major SpH whereas eggs have seven major histone variants, distinct from each other, with an electrophoretic behaviour consistent with histones but a different amino acid composition. The total complement of histone variants found in zygotes collected at amphimixis is identical with that found in unfertilized eggs, suggesting that SpH should be lost before that time.(ABSTRACT TRUNCATED AT 250 WORDS)

Even though the proto-oncogene c-Ki-ras is found to be amplified and overexpressed in the malignant Y-1 mouse corticoadrenal cell line, evidence for a causal relationship between c-Ki-ras overexpression and malignancy is still lacking. Such evidence is presented in this report. Amplified c-Ki-ras was found in both sublines of Y-1 cells that carry DM (double minute) or HSR (homogeneously stained region) chromosomes. Y-1 normal revertants did not display c-Ki-ras amplified sequences. Spontaneous retransformation of the normal revertants yielded anchorage-independent non-tumorigenic cells in which c-Ki-ras was not amplified. We propose that c-Ki-ras amplification is required to maintain the malignant state of Y-1 cells. Constitutive expression of poly A RNA homologous to viral sequences were detected in all sublines of Y-1 cells (malignant or normal revertants) by Northern hybridization using probes derived from the pFBJ-2 provirus. Rapid induction of c-fos proto-oncogene mRNA and late reduction in the levels of poly A+ viral transcripts resulted from ACTH treatment of Y-1 cells. However, ACTH treatment did not change c-Ki-ras mRNA levels.

The expression of the rat angiotensin-II receptor has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the brain and adrenal gland was injected into oocytes, and the expression of the receptor in the oocyte plasma membrane was assayed by measuring the change in membrane potential in the presence of angiotensin-II. Expression of the angiotensin-II receptor was detected 1.0-1.5 days after messenger RNA (mRNA) injection, and the degree of membrane depolarization was proportional to the amount of mRNA injected. Ca+2 channel blockers inhibited the angiotensin-II-induced depolarization. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce angiotensin-II depolarization. The mRNA encoding the angiotensin-II receptor was found in a single fraction of 4.4 kilobases.

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497. The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined. None of the base substitutions of C1400 were strongly inhibitory. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects of tRNA binding were variable. The only alteration to block all ribosomal function was the deletion of G1401. The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal. The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.

Early and transient expression of proto-oncogenes c-fos and c-myc is involved in the mitogenic response to PDGF (platelet-derived growth factor). We used DNA-mediated transfection to approach the role played by these genes in cell growth control by PDGF and in growth deregulation (neoplasia). Cloned pFBJ-2 (v-fos) and glucocorticoid-inducible mouse c-myc were co-transfected with a neo genetic marker to allow a neutral selection on the basis of resistance to the neomycin derivative geneticin G418. pFBJ-2 transfection was found to interfere with the number of G418-resistant (G418r) colonies. By using a v-fos-deleted pFBJ-2 construct, the deleterious effect was attributed to v-fos coding sequences. Cellular fos gene disruption, by homologous recombination with exogenous v-fos, is proposed as the basis for the deleterious effect. Co-transfection with MMTV-H3-c-myc effectively counteracts the negative effects of v-fos. Different from the parental line or single myc or fos transfectants, double myc/fos transfectants are morphologically transformed. Double transfectants still retain the PDGF requirement for growth in monolayer cultures.

Rotavirus are segmented double stranded RNA viruses with a double protein capsid around a central core. The virus replicates in the cell cytoplasm. After infection, eleven mRNAs are transcribed from the viral genome. To characterize further the infection cycle, viral polypeptide synthesis and RNA replication were studied using labelled precursors. The involvement of nonstructural polypeptides NS34 and NS35 was determined by the kinetics of the appearance of viral polypeptides in infected cells. Experiments in which cycloheximide was used showed that the synthesis of both polypeptides was required to begin RNA replication. The isolation of subviral particles at 8 hours postinfection indicates that there is a particle containing the nonstructural polypeptides and the structural polypeptides VP1, VP2, and VP6 that seem to be able to transcribe the viral genome to produce other RNA species. The results suggest that there is a core-like particle similar to one obtained in vitro which upon the addition of VP6 is able to transcribe the virus genome. This seems to indicate that core-like particles may alter their specificity for plus or minus RNA synthesis depending upon the polypeptides that interact with it. The interaction between VP6 and the viral core was analyzed by means of antibodies raised against the viral core and VP6. The results suggest that VP6 contains a specific binding site to the core complex and this interaction allows the synthesis of mRNA.