A novel sex-specific DNA marker in Columbidae birds

That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.     

Folding stability modulation of the villin headpiece helical subdomain by 4‐fluorophenylalanine and 4‐methylphenylalanine

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4‐fluorophenylalanine (Z) and 4‐methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4‐fluorophenylalanine and 4‐methylphenylalanine. Our results show that these two non‐natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 627–637, 2015.     

Infant Cancer in Taiwan: Incidence and Trends (1995-2009)

Background

Current information about cancer incidence patterns among infants in East Asia is rare. The objective of this study was to report the first population-based cancer surveillance of infants in Taiwan.

Methods

Cancer frequencies and incidence rates among subjects aged <1 year for the period 1995-2009 were obtained from the Taiwan Cancer Registry. Types of cancers were grouped according to the International Classification of Childhood Cancer. Rates and trends were analyzed by sex and disease groups and further compared with that of other countries.

Results

A total of 900 infants were diagnosed with cancers, giving an incidence rate of 250.7 per million person-years from 1995 to 2009. The male-to-female incidence rate ratio was 1.22. Overall, leukemias (56.3 per million) were the most common cancer, followed by germ cell neoplasms (43.2) and neuroblastomas (41.8). The incidence increased by 2.5% annually during the 15-year study period and was predominantly contributed by male infants (3.5%). Compared with other countries, the rate of hepatoblastoma in Taiwan was second to that from Beijing (China) and 2 to 5 times greater compared with the US, France, the North of England and Osaka (Japan). The rates of germ cell neoplasms were 2 to 4 times greater in Taiwan.

Conclusions

The current data suggests that cancer incidence rate among male infants was rising in Taiwan. The factors associated with higher rates of hepatoblastoma and germ cell neoplasms warrant further investigation on similar ethnic groups of different areas to elucidate the potential environmental impacts while controlling for race.     

The RssAB two-component signal transduction system in Serratia marcescens regulates swarming motility and cell envelope architecture in response to exogenous saturated fatty acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.     

Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1

The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis.     

Habitat-related mtDNA polymorphism in the stored-bean pest Callosobruchus chinensis (Coleoptera: Bruchidae)

The genetic diversity of populations of the azuki bean beetle, Callosobruchus chinensis (Linnaeus) from natural, pre-harvest and post-harvest sites, was investigated to understand population structure and gene flow. A 522-bp fragment of the mitochondrial gene COI was sequenced for eight populations of C. chinensisfrom Japan, Korea and Taiwan collected from different habitats. Six haplotypes were detected, one of which, U1, occurred most frequently and widely. The following hypotheses were tested as a cause of the wide distribution of haplotype U1; (i) topographical separation (by national boundaries), (ii) host plant species, and (iii) habitat type (natural, pre-harvest crop, or post-harvest storage). Categorization of collection sites by country or by host species did not yield differences in the occurrence of haplotype U1, but habitat type did. Populations utilizing cultivated post-harvest hosts that were mass stored were highly likely to be the common haplotype, whereas host plants in natural habitats away from agriculture were utilized by populations with locally characteristic haplotypes. Sampling of commercial beans for quarantine and export purposes indicated that gene flow in C. chinensis was largely unidirectional into Japan at the present time.     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding

pH-Dependent studies of the folding kinetics and stability of a set of His to Gln point mutants were used to characterize the denatured state and transition state ensembles for the C-terminal domain of the ribosomal protein L9 (CTL9). CTL9 contains three histidine residues, two of which, H106 and H134, are buried in the native state, while the third, H144, is more exposed. Comparison of the pH-dependent stability calculated using the Tanford-Wyman linkage relationship to the measured values demonstrates that the apparent pK(a) values of the three histidine residues are not significantly perturbed in the denatured state ensemble. Kinetic measurements show that mutation of H134 has a larger effect on the folding process than does mutation of H106 and H144. The Phi-value for H134 is significantly larger than the Phi-values for the other histidine residues, which are near zero at both pH 5.45 and pH 8.0. The Phi-value for H134 is higher, 0.55, at pH 8.0 than at pH 5.45, 0.39. At pH 5.45, H134 is protonated in the unfolded state but deprotonated in the native state, while at pH 8.0 it is deprotonated in both. There is an excellent linear relationship between stability (logK) and folding rates (logk(f)) over the range of pH 5-9 for all mutants. From these plots, the ratio of DeltaQ( not equal)/DeltaQ can be calculated for each mutant. DeltaQ( not equal) is the difference in the number of protons bound to the transition state and to the unfolded state, while DeltaQ represents the difference between folded and denatured state. The linear plots indicate that the relative position of the transition state ensemble as judged by DeltaQ( not equal)/DeltaQ is independent of pH. The linkage analysis is consistent with the Phi-value analysis, showing that H134 is the most critical contributor to the development of pH-dependent interactions, including desolvation effects in the transition state ensemble.