The int genes of bacteriophages P22 and λ are regulated by different mechanisms

Bacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII-dependent P_I promoter is responsible for λint gene expression. The only apparent counterpart to P_I in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called P_al, is active both in vivo and in vitro, and is dependent upon the P22 cII-like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 p_L promoter and the int gene. The newly determined sequence is densely packed with genes from the p_L direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (p_al) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in λ.     

Alterations in diaphragmatic oxidative and antioxidant enzymes in the senescent Fischer 344 rat.

We investigated age-related changes in antioxidant, glycolytic, beta-oxidation, and tricarboxylic acid cycle enzyme activity in the diaphragm and plantaris muscle of female Fischer 344 rats. Tissue samples from the costal and crural diaphragm and plantaris muscle were obtained from 30 animals in the following age groups: 1) 6 mo old (n = 10), 2) 26 mo old (n = 10), and 3) 30 mo old (n = 10). Aging had no effect (P greater than 0.05) on the activities of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HADH) in the costal or crural diaphragm. Similarly, no age-related differences existed (P greater than 0.05) in the crural diaphragm in lactate dehydrogenase (LDH) or glutathione peroxidase (GPX) activity. In contrast, the activities of LDH and GPX were significantly (P less than 0.05) higher in the costal diaphragm in the 30- than in the 6-mo old animals. In addition, the ratio of LDH to CS activity increased (P less than 0.05) as a function of age in the costal diaphragm. Conversely, the ratio of CS to GPX activity in the costal diaphragm was lower (P less than 0.05) in the 30- than in the 6-mo old animals. No significant (P greater than 0.05) age-related differences existed in LDH-to-CS or CS-to-GPX activity ratios in the crural diaphragm. Finally, aging resulted in a significant decrease (P less than 0.05) in the activities of LDH, CS, and HADH in the plantaris muscle. These data demonstrate that, unlike many hindlimb locomotor muscles, the oxidative capacity of the Fischer 344 rat diaphragm does not decrease in old age.     

Substrate specificity of Ty1 integrase.

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 requires the element-encoded integrase (IN) protein, which is a component of cytoplasmic virus-like particles (VLPs). Using purified recombinant Ty1 IN and an oligonucleotide integration assay based on Ty1 long terminal repeat sequences, we have compared IN activity on substrates having either wild-type or altered donor ends. IN showed a marked preference for blunt-end substrates terminating in an A:T pair over substrates ending in a G:C pair or a 3' dideoxyadenosine. VLP activity on representative substrates also showed preference for donor strands which have an adenosine terminus. Staggered-end substrates showed little activity when nucleotides were removed from the end of the wild-type donor strand, but removal of one nucleotide from the complementary strand did not significantly diminish activity. Removal of additional nucleotides from the complementary strand reduced activity to minimal detection levels. These results suggest that the sequence specificity of Ty1 IN is not stringent in vitro. The absence of Ty1 IN-mediated 3' dinucleotide cleavage, a characteristic of retroviral integrases, was demonstrated by using selected substrates. In addition to the forward reaction, both recombinant IN and VLP-associated IN carry out the reverse disintegration reaction with long terminal repeat-based dumbbell substrates. Disintegration activity exhibits sequence preferences similar to those observed for the forward reaction.     

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.     

Genetic analysis of yeast RAS1 and RAS2 genes

We present a genetic analysis of RAS1 and RAS2 of S. cerevisiae, two genes that are highly homologous to mammalian ras genes. By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes. However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth. We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles.     

CO bond angle changes in photolysis of carboxymyoglobin

Previous studies [Chance, B., Fischetti, B., & Powers, L. (1983) Biochemistry 22, 3820-3829] of the local structure changes around the iron in carboxymyoglobin on photolysis at 4 K revealed that the iron-carbon distance increased approximately 0.05 A but was accompanied by a lengthening of the iron-pyrrole nitrogen bonds of the heme (approximately 0.03 A) that was not as large as that found in the deoxy form. Further analysis of these data together with comparison to model compounds indicates that the Fe-C-O bond angle in carboxymyoglobin is bent (127 +/- 4 degrees), having a structure identical, within the error, with the "pocket" porphyrin model compound FePocPiv(1-MeIm)(CO) [Collman, J. P., Brauman, J. I., Collins, T. J., Iverson, B. L., Lang, G., Pettman, R., Sessler, J. L., & Walters, M. A. (1983) J. Am. Chem Soc. 105, 3038-3052]. On photolysis, this angle decreases by 5-10 degrees. In addition, correlation is observed between the increase in the length of the Fe-C bond and the decrease of the Fe-C-O angle. These results suggest that the rate-limiting step in recombination is the thermal motion of CO in the pocket to achieve an appropriate bonding angle with respect to the iron. These changes constitute the first molecular picture of the photolysis process, as well as the structure of the geminate state, and are important in clarifying nuclear tunneling parameters.     

Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase)

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.