Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin

The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha.     

Spectrophotometric assay for ornithine decarboxylase

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.     

Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.     

Epithelial impedance analysis in experimentally induced colon cancer.

Epithelial impedance analysis was used to measure the alterations in resistance of the large bowel in a murine model of large bowel cancer. The technique was able to resolve the epithelial resistance from the total resistance of the bowel wall. A progressive decrease in resistance of the bowel epithelium occurs during carcinogenesis induced with dimethyhydrazine. About a 21% decrease in epithelial resistance from 22.0 +/- 1.3 omega.cm-2 to 17.5 +/- 1.1 omega cm-2 (p less than 0.025) was observed after 20 wk of carcinogen administration. The sensitivity of the technique in detecting altered epithelial resistance in premalignant bowel mucosa was improved by examining the impedance profile in a sodium-free Ringer's solution where the epithelium of control colons had a resistance of 24.4 +/- 1.8 omega.cm-2 compared with 19.0 +/- 1.1 omega.cm-2 (p less than 0.02) in colons from animals treated for only 4 wk with the carcinogen. Epithelial impedance analysis would seem to be a sensitive technique capable of identifying changes in the electrical properties or the large bowel early in disease states.     

The Putative Oncogene Pim-1 in the Mouse: Its Linkage and Variation among t Haplotypes

Pim-1, a putative oncogene involved in T-cell lymphomagenesis, was mapped between the pseudo-alpha globin gene Hba-4ps and the alpha-crystallin gene Crya-1 on mouse chromosome 17 and therefore within the t complex. Pim-1 restriction fragment variants were identified among t haplotypes. Analysis of restriction fragment sizes obtained with 12 endonucleases demonstrated that the Pim-1 genes in some t haplotypes were indistinguishable from the sizes for the Pim-1b allele in BALB/c inbred mice. There are now three genes, Pim-1, Crya-1 and H-2 I-E, that vary among independently derived t haplotypes and that have indistinguishable alleles in t haplotypes and inbred strains. These genes are closely linked within the distal inversion of the t complex. Because it is unlikely that these variants arose independently in t haplotypes and their wild-type homologues, we propose that an exchange of chromosomal segments, probably through double crossingover, was responsible for indistinguishable Pim-1 genes shared by certain t haplotypes and their wild-type homologues. There was, however, no apparent association between variant alleles of these three genes among t haplotypes as would be expected if a single exchange introduced these alleles into t haplotypes. If these variant alleles can be shown to be identical to the wild-type allele, then lack of association suggests that multiple exchanges have occurred during the evolution of the t complex.     

The Influence of Nonautonomous P Elements on Hybrid Dysgenesis in Drosophila melanogaster

An inbred line of the M' strain Muller-5 Birmingham was studied for its abilities to affect P-M hybrid dysgenesis. This strain possesses 57 P elements, all of which are apparently defective in the production of the P transposase. In combination with transposase-producing elements, these nonautonomous elements can enhance or diminish the incidence of hybrid dysgenesis, depending on the trait that is studied. Dysgenic flies that have one or more paternally-derived chromosomes with these elements partially repress the instability of the P element insertion mutation, snw; however, such flies have elevated frequencies of another dysgenic trait, GD sterility, and also show distorted segregation ratios. An explanation is presented in which all of these phenomena are unified as manifestations of the kinetics of P element activation in the germ line. The progeny of Muller-5 Birmingham females exhibit partial repression of both snw instability and GD sterility. This repression appears to involve a factor that can be transmitted maternally through at least two generations. This mode of repression therefore conforms to the pattern of inheritance of the P cytotype, the condition that brings about nearly total repression of P element activity in some strains. Models in which this repression could arise from the nonautonomous P elements of Muller-5 Birmingham are discussed.     

A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication.