Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Probability of paternity exclusion and the number of loci needed to determine the fathers in a troop of macaques

We calculated the probability of paternity exclusion (P) in 6 troops of rhesus and Japanese macaques housed in open enclosures and 68 wild troops of Japanese, crab-eating, and toque macaques using 33 genetic loci which encoded the blood protein variations detected by electrophoretic techniques. In the open enclosures, especially of rhesus troops, we obtained a fairly high probability of paternity exclusion and succeeded in determining the fathers of offspring. However, we found significant differences between the observed and calculated probabilities in most of the troops. These differences were ascribed to a situation whereby the Hardy-Weinberg equilibrium had not been attained in the troops and/or the numbers of variable loci were too small. In the wild troops of Japanese, crab-eating, and toque macaques, the means ofP were 0.2274 (0.0192–0.5017), 0.4635 (0.1676–0.7151), and 0.7382 (0.6266–0.7954), respectively. We also estimated the number of loci needed to determine the fathers of offspring with a probability of 0.8 assuming that ten males were present in the troop. The estimated number was about 13.5 times, 5 times and 1.8 times the number of loci examined on average in the troops of Japanese, crab-eating and toque macaques, respectively. This means that determination of most of the fathers of offspring in wild troops of these macaques, even of toque macaques which have a rather high probability of paternity exclusion, is difficult so long as we employ only electrophoretic techniques.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.