Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.     

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs. We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro. Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms. Pure Y-structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains. One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent. The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure. Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures. The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA.     

Evolution of a New Gene Substituting for the leuD Gene of Salmonella typhimurium: Characterization of supQ Mutations

Alpha-isopropylmalate isomerase, the second specific enzyme in the biosynthesis of leucine, is coded for by two genes, leuC and leuD. Leucine auxotrophs, harboring leuD mutations including a deletion of the entire leuD gene, revert to leucine prototrophy owing to mutations at a locus, supQ, substantially distant to the leucine operon. A large number of independently isolated supQ mutations were characterized. A significant increase in the spontaneous frequency of supQ mutations was found after mutagenesis with 2-aminopurine, N-methyl-N′-nitro-N-nitrosoguanidine, diethyl sulfate, and nitrous acid. The supQ function in most of these strains is temperature sensitive, resulting in more efficient suppression with decreasing temperature. At higher temperatures, the supQ limits the growth rate of leuD supQ mutant strains. All supQ mutations are co-transducible with proA and proB, with co-transduction frequencies ranging from 5.4 to 99.9% for different supQ mutations. Many supQ mutations were isolated, especially after nitrous acid mutagenesis, that had acquired a simultaneous proline requirement. The data support the idea of two genes, supQ and newD, whose protein products form a complex. The newD gene product, without any genetic alteration, is capable of substituting for the missing leuD protein. However, mutations in the supQ gene (point mutations or deletions) are necessary to make the newD protein available, which is normally tied up in a complex with the supQ protein.     

High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells

Hybrids were made between rat glioma X mouse neuroblastoma cell lines and were examined for the specific activities of choline acetyltransferase and acetylcholinesterase. The specific activities of choline acetyltransferase of the hybrids were as high as those in normal brain, whereas neither parent line showed appreciable activities. The electrical excitability of the neuroblastoma cells was retained in the hybrids.     

Influence of dietary nicotine and colony source ofManduca sexta [Lepidoptera: Sphingidae] on its suitability as a host ofCotesia congregata [Hymenoptera: Braconidae]

Larval tobacco hornworms,Manduca sexta (L.), of 2 different colonies were exposed to parasitism by the gregarious endoparasitoid,Cotesia congregata (Say). A comparison was made of parasitoid larval, pre-pupal, and pupal mortality, female and male dry weight and larval development time. In general, “Maryland” hornworms were more suitable hosts than “North Carolina” hornworms. Although the presence of dietary nicotine increased parasitoid mortality in individuals reared from hornworms of both colonies, the effect was more severe among individuals parasitizing the North Carolina hornworms. Scientific contribution No. 8125, article No. A-5066 of the Maryland Agricultural Experiment Station, Department of Entomology.