Polymorphism of alkaline ribonuclease in the leucocytes of Black-and-White cattle

Studies were carried out on the polymorphism of alkaline ribonuclease in Black-and-White cattle in the north-eastern region of Poland. Leucocytes isolated from peripheral blood were used. Three phenotypes were defined, consisting of the combination of two alleles. The gene frequency was: q⁴= 0,7082, q^B= 0,2918. In calves the frequency of allele RNA-ase^B and number of RNA-ase^A/ RNA -ase^B heterozygotes were higher than in adult animals.     

Abnormal segregation of prion protein octapeptide-repeat alleles in cattle

The study was conducted on full-families of Black-and-White cattle obtained as 25 AI sire families and 355 cows, as well as their progenies, mostly heifers at the age of 1-3 months. The sire group was composed by the casual qualification of 10 PRNP 6/6 and 15 PRNP 6/5 individuals on the basis of accessible young progenies. The randomly selected group of cows is characterised by a very high frequency of PRNP 6/6 (74.9%), followed by lower frequency of PRNP 6/5 (24.5%) and a very low frequency of PRNP 5/5 genotype (0.6%). The progenies represent all expected genotypes, such as: PRNP 6/6 (60.5%), PRNP 6/5 (35.8%) and PRNP 5/5 (3.7%), respectively. Taking into consideration the genotypes of parents and progenies, the segregation of PRNP 6 and PRNP 5 alleles was analysed. Results of the non-informative mating variant of male symbol PRNP 6/6 x female symbol PRNP 6/6 (n = 87) are affected by the PRNP 6/6 progeny genotype in all cases. Subsequently, the results of mating variants male symbol PRNP 6/6 x female symbol PRNP 6/5 (n = 29) and male symbol PRNP 6/5 female symbol PRNP 6/6 (n = 179) showed statistically non-significant differences in both above-mentioned alternations. The progeny group related from male symbol PRNP 6/5 x female symbol PRNP 6/5 parental mating obtained fully informative and most valuable results based on the presented research concept. In the common group of 58 calves, the genotype PRNP 6/6 is represented by 26 individuals (44.8%), PRNP 6/5 - by 19 individuals (32.8%) and PRNP 5/5 - by 13 individuals (22.4%). Therefore, the theoretical genotype rate (25% : 50% : 25%) is drastically deformed and the differentiation between the observed and expected numbers of animals is statistically highly significant (chi(2) = 12.72; 2 df.). These differences are affected by two times higher PRNP 6/6 homozygous (chi(2) = 9.12; 1 df.) and responsively by the low number of PRNP 6/5 heterozygous animals (chi(2) = 3.45; 1 df.). Further investigations are carried out to explain the genetic determination of abnormal PRNP octa-peptide repeat allele segregation, which suggests possible lethal cis-trans linkage effects.     

Circular zymogens of human ribonuclease 1

The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 10⁴‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.     

Sequence variants in the bovine nucleophosmin 1 gene, their linkage and their associations with body weight in native cattle breeds in China

The nucleophosmin (nucleolar phosphoprotein B23, numatrin) gene (NPM1, previously known as nucleophosmin/nucleoplasmin family, member 1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in cell growth and homeostasis. Seven sequence variants (SVs) were identified in the coding region of bovine NPM1, five of which were in complete linkage disequilibrium. Eight different haplotypes were identified, of which two major haplotypes have a frequency of 23.2% and 20.4%. Three SVs were significantly associated with body weight in the Nanyang population as analysed at different ages. No significant association was detected between 18 combined genotypes and body weight at five different ages. Our results suggest that some polymorphisms in NPM1 are associated with body weight at some ages and may be used as candidates for marker-assisted selection and management in beef cattle breeding programmes.     

Fluorescence assay for the binding of ribonuclease A to the ribonuclease inhibitor protein

Ribonuclease A (RNase A) and the ribonuclease inhibitor protein (RI) form one of the tightest known protein-protein complexes. RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells. Herein, a new and facile assay is described for measuring the equilibrium dissociation constant (K(d)) and dissociation rate constant (k(d)) for complexes of RI and RNase A. This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI. To allow time for equilibration, the assay is most readily applied to those complexes with K(d) values in the nanomolar range or higher. Using this assay, the value of K(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 +/- 0.03 nM. In addition, the value of K(d) was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 +/- 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A. Finally, the value of k(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 +/- 0.4) x 10(-3) s(-1) by monitoring the increase in fluorescence intensity upon dissociation. This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.     

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor.

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.     

Frequencies of genes for coat colour and horns in Nordic cattle breeds

Gene frequencies of coat colour and horn types were assessed in 22 Nordic cattle breeds in a project aimed at establishing genetic profiles of the breeds under study. The coat colour loci yielding information on genetic variation were: extension, agouti, spotting, brindle, dun dilution and colour sided. The polled locus was assessed for two alleles. A profound variation between breeds was observed in the frequencies of both colour and horn alleles, with the older breeds generally showing greater variation in observed colour, horn types and segregating alleles than the modern breeds. The correspondence between the present genetic distance matrix and previous molecular marker distance matrices was low (r = 0.08 – 0.12). The branching pattern of a neighbour-joining tree disagreed to some extent with the molecular data structure. The current data indicates that 70% of the total genetic variation could be explained by differences between the breeds, suggesting a much greater breed differentiation than typically found at protein and microsatellite loci. The marked differentiation of the cattle breeds and observed disagreements with the results from the previous molecular data in the topology of the phylogenetic trees are most likely a result of selection on phenotypic characters analysed in this study.     

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.     

Bayesian segregation analysis of milk flow in Swiss dairy cattle using Gibbs sampling

Segregation analyses with Gibbs sampling were applied to investigate the mode of inheritance and to estimate the genetic parameters of milk flow of Swiss dairy cattle. The data consisted of 204 397, 655 989 and 40 242 lactation records of milk flow in Brown Swiss, Simmental and Holstein cattle, respectively (4 to 22 years). Separate genetic analyses of first and multiple lactations were carried out for each breed. The results show that genetic parameters especially polygenic variance and heritability of milk flow in the first lactation were very similar under both mixed inheritance (polygenes + major gene) and polygenic models. Segregation analyses yielded very low major gene variances which favour the polygenic determinism of milk flow. Heritabilities and repeatabilities of milk flow in both Brown Swiss and Simmental were high (0.44 to 0.48 and 0.54 to 0.59, respectively). The heritability of milk flow based on scores of milking ability in Holstein was intermediate (0.25). Variance components and heritabilities in the first lactation were slightly larger than those estimates for multiple lactations. The results suggest that milk flow (the quantity of milk per minute of milking) is a relevant measurement to characterise the cows milking ability which is a good candidate trait to be evaluated for a possible inclusion in the selection objectives in dairy cattle.     

Allelic polymorphism in Arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease

Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.Part of this work has been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.).     

Chromosome markers in cattle

Summary Giemsa banding in cattle chromosomes enables the demarcation of both centromeric areas as pale regions and banding patterns along the chromosome arms. These are valuable in identifying all the chromosomes of a given karyotype. A high degree of intra- and inter-individual variation in the size of the centromeres was observed. This variation is useful for the identification of each individual and provides a broad base of chromosome markers for cattle breeding.     

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1)

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.     

Chromosomal assignment of six muscle-specific genes in cattle

Six genes expressed in skeletal or smooth muscle were assigned to bovine chromosomes using rodent, human or bovine cDNA probes. Myogenic determination factor (MYOD1) was 100% concordant with Bos taurus chromosome (BTA) 15, and myogenin (MYOG) was 95% concordant with BTA 16. Smooth muscle caldesmon (CALD1) and the skeletal muscle chloride channel gene (CLCN1) were 100% concordant with BTA 4. Myogenic factor 5 (MYF5) was 90% concordant with BTA 5; this assignment was confirmed by fluorescence in situ hybridization of a bovine genomic MYF5 probe to BTA 5 band 13 and the homologous band on river buffalo 4q. In some metaphases, specific hybridization signals were also observed on BTA 15 band 23, and the equivalent river buffalo homologue, with the MYF5 genomic probe. Because MYOD1 and MYF5 share both nucleotide and functional homology and because MYOD1 was mapped in somatic cell hybrids to BTA 15, we suggest that MYOD1 may be located at BTA 15 band 23. Herculin/myogenic factor 6 (MYF6) was assigned indirectly to BTA 5 by the hybridization of MYF5 and MYF6 probes to the same Hin dIII fragment in bovine genomic DNA. The assignment of MYF6 to BTA 5 is consistent with the tandem arrangement of MYF5 and MYF6 in human, mouse and chicken, where these tightly linked genes are separated by < 6·5 kb of DNA.     

Serum alkaline phosphatase polymorphism in pigs1

Genetic variants of serum alkaline phosphatase were studied by the method of starch gel electrophoresis in the Zlotnicka Pstra breed of pigs. Two regions of alkaline phosphatase migration were observed. A single fraction in region I and four different phenotypes: AB, B, BC and BD in region II, were found. For AB, B and BC phenotypes the genetic control by three alleles AkpA, AkpB and AkpC in suggested. The observed segregation ratios in some cases deviated significantly from the expected ones.     

Concentration dependence of the effect ofBacillus intermedius ribonuclease on the yeastSaccharomyces cerevisiae

Bacillus intermedius RNase added at a low concentration (0.001 μg/ml) stimulated yeast growth, while a high RNase concentration (1500 μg/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.     

Detection of genes influencing economic traits in three French dairy cattle breeds

A project of QTL detection was carried out in the French Holstein, Normande, and Montbéliarde dairy cattle breeds. This granddaughter design included 1 548 artificial insemination bulls distributed in 14 sire families and evaluated after a progeny-test for 24 traits (production, milk composition, persistency, type, fertility, mastitis resistance, and milking ease). These bulls were also genotyped for 169 genetic markers, mostly microsatellites. The QTL were analysed by within-sire linear regression of daughter yield deviations or deregressed proofs on the probability that the son receives one or the other paternal QTL allele, given the marker information. QTL were detected for all traits, including those with a low heritability. One hundred and twenty QTL with a chromosome-wise significance lower than 3% were tabulated. This threshold corresponded to a 15% false discovery rate. Amongst them, 32 were genome-wise significant. Estimates of their contribution to genetic variance ranged from 6 to 40%. Most substitution effects ranged from 0.6 to 1.0 genetic standard deviation. For a given QTL, only 1 to 5 families out of 14 were informative. The confidence intervals of the QTL locations were large and always greater than 20 cM. This experiment confirmed several already published QTL but most of them were original, particularly for non-production traits.     

Research on association between variants and haplotypes of TRPV1 and TRPA1 genes with growth traits in three cattle breeds

The objective of this study was to examine the association of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) genes polymorphisms with growth traits in three Chinese cattle breeds (Jiaxian red cattle, Qinchuan cattle and Luxi cattle). Through experiments we identified three single nucleotide polymorphisms (SNPs) in these three cattle breeds TRPV1 and TRPA1 genes using PCR-SSCP, (forced) PCR-RFLP methods. Three of these polymorphisms are all synonymous mutation which includes (NW_003104493.1: 30327 C?>?T), (NW_003104493.1: 33394 A?>?G) and (NW_003104493.1: 38471?G?>?A) are in exons. The other three polymorphisms are located at 3'UTR. Furthermore, we evaluated the haplotype frequency and the statistical analyses indicated that these SNPs of TRPV1 and TRPA1 genes were associated with bovine body height, body length, waist angle width, hucklebone width, cross ministry height, chest qingwidth (p?<?0.05) and recommendation height, cannon circumference (p?<?0.01) of Qingchuan cattle; body length, waist angle width (p?<?0.05) of Jiaxian red cattle; body weight, Body length, cannon circumference, chest circumference (p?<?0.05) and body height (p?<?0.01) of Luxi cattle. Our result confirms the polymorphisms in the TRPV1 and TRPA1 genes are associated with growth traits that may be used for marker-assisted selection (MAS) in three beef breeds programs.     

Characterization of a ribonuclease from Anacystis nidulans infected with cyanophage as-1

In Anacystis nidulans the ribonuclease (RNase) activity is very low but is greatly increased upon phage-infection. A RNase was isolated and purified over 300-fold from A. nidulans cells infected by cyanophage AS-1. The enzyme did not attack single- or double-stranded DNA, was inactive on p-nitrophenyl phosphate or bis-p-nitrophenyl phosphate as substrates, and had neither 3′- nor 5′-nucleotidase activity. The approximate MW of the enzyme was 12000. Maximal enzyme activity was at pH 7.5. No absolute requirement for metal ions was observed, but Fe³⁺ stimulated and Co²⁺ and Ni²⁺ inhibited enzyme activity. The enzyme is an endonuclease which, upon exhaustive hydrolysis, produces mainly oligonucleotides (average chain-length: 3) with 3′-P termini. Analysis of the base composition of these oligonucleotides and determination of their 3′-terminal nucleosides, together with the investigation of the rate of hydrolysis of synthetic polyribonucleotides, have shown that the enzyme has a relative specificity for uridylic acid.