Poly(ADP-ribosylation) of atypical CS histone variants is required for the progression of S phase in early embryos of sea urchins.

The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.     

Differentiation of New Metastatic Variants of B16 Melanoma Under Different Culture Conditions

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow A^y/a, albino c/c, and C⁺/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5-S-CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C⁺/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.     

Studies on the properties of the variants of gamma-glutamyl transpeptidase in human urine

Both the high molecular weight and the low molecular weight variants of urinary Y-glutamyl transpeptidase, displayed transpeptidase (pH optimum 8.6) and autotrans-peptidase (pH optimum 9.4) activities. Iodoacetamide inhibited the transpeptidase activity more efficiently than the autotranspeptidase activity with respect to both variants of Y-glutamyl transpeptidase. The high molecular weight form utilized L-glutamine as a better acceptor than L-cystine during the transpeptidation reaction whereas the reverse was the case with the low molecular weight variant. While phenylmethylsulphonyl fluoride-treated enzymes retained full activitiesper se, addition of maleic acid to the modified enzyme was found to inhibit the catalytic activities indicating a maleic acid-induced conformational change of the modified enzyme.     

Nucleotide sequence of Rattus norvegiens mitochondrial DNA that includes the genes for tRNAile, tRNAgln and tRNAf-met

The nucleotide sequence of a segment of mtDNA from Rattus norvegiens (rat) which contains the genes for tRNA^ile, tRNA^gl and tRNA^f-met has been determined. A detailed comparison has been made between this sequence and the corresponding sequences of mouse, human and bovine mtDNAs with regard to the primary and secondary structure of the tRNA genes, the regions connecting the tRNA genes, and the regions flanking the tRNA genes which code for the carboxyl terminus of URF-1 and the amino terminus of URF-2. No differences were found in the nucleotide sequences of the genes for tRNA^ile, tRNA^gln and tRNA^f-met in mtDNAs from three different female lines of rats (SASCO-1, SASCO-2 and Wild-UT) that differ by substitutions of 0.8% to 1.8% of their total nucleotides.     

Structures of cytokinins influence synergistic production of ethylene

Twenty-five naturally occurring cytokinins and structurally related compounds were tested for their ability to promote ethylene production synergistica     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

Biochemical and immunological studies of three genetic variants of 3-phosphoglycerate kinase 2 from the mouse

Three electrophoretic variants of 3-phosphoglycerate kinase 2 (PGK-2A, PGK-2B, and PGK-2C) were purified from DBA/2J, C3H/HeJ, and C57L/J mice, respectively. PGK-2C exhibits only 2% of the specific activity of PGK-2A and PGK-2B in the reaction leading to the formation of 1,3-diphosphoglycerate. Compared to PGK-2A and PGK-2B, PGK-2C exhibits broader coenzyme specificity and lower K_ms for substrate and coenzymes. Incubation at 45C revealed that PGK-2B is more heat stable than either PGK-2A or PGK-2C. Enzyme immunoinactivation and double immunodiffusion studies showed that mice carrying any one of these three PGK-2 alleles have similar amounts of proteins for PGK-1 and PGK-2 in testes. The results of these studies suggest that low PGK-2C activity in C57L/J mice is a result of a structural rather than a regulatory gene mutation.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16–010 cells grew in circular in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melainin pigment (B16-010 < B16-05 < B16-01 ? B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.     

Natural Variants of the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Twenty different isolates of the cellular slime mold Acrasis rosea, obtained from diverse sources and geographic regions, were studied to determine similarities and differences in their development and structure in culture and their sensitivity or resistance to selected chemicals incorporated into the culture media. Six different classes of fruiting were defined based on the size, distribution, and type of sorocarps formed on the yeast, Rhodotorula, streaked on agar. In the course of these studies a significant mutant, NC-18V (variant), developed spontaneously from the wild type, normal parent strain NC-18N. The mutant differed considerably from all other Acrasis isolates, appeared several times in purified parental cultures, and represents the first laboratory derived variant of A. rosea to be described. Purified strains of the variant (V) and normal (N) cultures were obtained by single-spore isolation. Normal and variant amebae were mixed in ratios of 10:1 and 100:1 (N:V) and spore and stalk cells were selected from different sorocarps in various regions of the culture plate for analysis. The results of these selection experiments clearly indicate that the individual variant amebae have increased migratory ability and that they develop smaller, morphologically different, and more numerous sorocarps that form at distances further from the food source than NC-18N. Some isolates of Acrasis no longer were able to fruit and were classified as “non-fruiters” and a few other isolates formed only a few, small sorocarps on rare occasions. These isolates were mixed together in various combinations of 2 and 3 to screen for cell interaction, but no synergism contributing to fruiting was found. Although fruiting of many A. rosea isolates was inhibited by exposure to continuous light or constant darkness, some “escape”fruiting was noted in certain isolates even when small inocula were used. Single spore isolates of these escape fruiters still fruited in continuous light or dark, but fruiting was always greatly enhanced by a routine 12 hr light : 12 hr dark incubation cycle. It was shown by biochemical studies that actidione, crystal violet, malachite green, ethyl violet, and 5-fluorodeoxyuridine selectively killed some isolates and permitted a classification of isolates as either sensitive or resistant. In a further study of cell interaction between 2 different sets of Acrasis isolates with contrasting biochemical and morphologic markers the formation of neotypes or recombinants could not be demonstrated. The results of this study clearly indicate the existence of significant variation in A. rosea and the potential for application of these differences to developmental studies.