Allelic Variation in the Porcine MYF5 Gene Detected by PCR–SSCP

The MYF5 gene has been reported to be integral to muscle growth and development, and hence it has been considered as a candidate gene for meat selection programs in pig. To ascertain whether there was variation in the porcine MYF5 gene, we have developed a method of PCR–single-strand conformational polymorphism (PCR–SSCP) analysis. In this study, two coding regions of the MYF5 gene were investigated. Four unique SSCP patterns were detected in exon 1 and three patterns were identified in exon 3. Two SNPs detected in exon 1 led to a non-synonymous alanine/proline substitution. A nucleotide change in exon 3 did not affect the amino acid sequence. Five extended haplotypes were observed across the two regions. The variation detected in this study might underpin the development of gene markers for improved muscle growth in pig breeding.     

Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow

The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P?MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.     

Cloning and tissue distribution of Leptin mRNA in the pig∗

Abstract

The sequence of the pig ob cDNA, which codes for the protein leptin, has been determined by screening a pig adipose cDNA library with an RT‐PCR amplified cDNA fragment of this gene. The 501 bp ob cDNA has 89% identity to the human ob cDNA, 92% identity to the bovine ob cDNA, 84% identity to the mouse ob cDNA and 84% identity to the rat ob cDNA. At the amino acid level, pig leptin which codes for a protein with a predicted molecular weight of 18,661‐dalton, has 86% identity to human leptin, 93% identity to bovine leptin, 84% identity to rat leptin and 84% identity to mouse leptin. RT‐PCR screening of RNA isolated from pig adipose, skeletal muscle, cardiac muscle, pancreas, stomach, kidney, spleen and jejunum detected ob mRNA only in adipose tissue; Northern blots with an ob cDNA probe identified a 4.0 kb species in adipose tissue. The conservation of sequence and expression pattern of leptin in the pig reported here indicates that as in other species, this protein likely plays an important role in controlling food intake and fat deposition in the pig.     

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.     

Seedling assemblages and the alternative successional pathways experienced by Atlantic forest fragments

Background: Habitat loss and fragmentation have been argued to drastically alter the composition of tree assemblages inhabiting small forest fragments but the successional trajectory experienced by such edge-affected habitats remains controversial.

Aims: Here we examine whether small fragments (3.4–91.2 ha) support seedling assemblages more similar to those in 10–70-year-old secondary forests than to those in mature forests, in order to infer to what extent fragments move toward early successional systems.

Methods: Using 59 0.1-ha plots distributed in a fragmented landscape of Brazilian Atlantic forest, we evaluated species richness and functional and taxonomic composition of seedling assemblages in 20 small forest fragments, 19 stands of secondary forest and 20 stands of mature forests in the interior of an exceptionally large fragment (ca. 3500 ha).

Results: Small fragments presented the least species-rich seedling assemblages (17.2 ± 5.7 species), followed by secondary (22.5 ± 5.3), and mature forest (28.4 ± 5.3). Small fragments had seedling assemblages with functional and taxonomic composition more similar to those in secondary than in mature forest. Small fragments had a greater relative richness and abundance of pioneer trees (ca. 40% more), vertebrate-dispersed (6–25%), and those bearing medium-sized seeds (30–70%), while large-seeded species and individuals were reduced (>50% decrement) in comparison to seedling assemblages in mature forest.

Conclusions: By comparing seedlings across a wide range of successional habitats we offer evidence that small forest fragments are experiencing an alternative successional pathway towards an early-successional system with reduced plant diversity.     

一株嗜酸硫杆菌M4-422-6的分离及其全基因组测序和比较基因组学分析

【目的】开展具有硫氧化能力的嗜酸硫杆菌属(Acidithiobacillus)的分离及其比较基因组学分析,不仅可以丰富硫氧化细菌菌种资源,而且有助于加深理解嗜酸硫杆菌的分子进化与生态适应机制。【方法】利用以硫代硫酸钠为唯一能源的培养基分离具有硫氧化能力的细菌;利用Illumina HiSeq X和Oxford Nanopore测序平台对一株嗜酸硫杆菌M4-422-6进行全基因组测序;利用相关生物信息学分析软件对原始数据进行组装和基因组注释,并与一株亲缘关系最近的菌株Igneacidithiobacillus copahuensis VAN18-1进行比较基因组学分析。【结果】分离获得一株具有硫氧化能力的嗜酸硫杆菌M4-422-6。基因组注释结果显示,菌株M4-422-6基因组由1个染色体和2个质粒组成,基因组大小为2 917 823 bp,G+C含量为58.54%,共编码2 925个蛋白。16S rRNA基因和基因组系统发育树显示,菌株M4-422-6代表嗜酸硫杆菌属的一个潜在新种。功能基因注释结果显示,菌株Acidithiobacillus sp. M4-422-6拥有与菌株特性相关的众多基因,包括硫氧化相关基因、CO₂固定相关基因和耐酸相关基因。比较基因组学分析发现,虽然菌株M4-422-6与VAN18-1的亲缘关系最近,但两者仍拥有众多的差异基因,主要包括噬菌体抗性相关基因和移动元件编码基因。【结论】菌株M4-422-6代表嗜酸硫杆菌属的一个潜在新种,该菌株具有同种内菌株所不具有的特有基因,并据此推测嗜酸硫杆菌种内分化可归因于对特定生态位的适应。     

Genome sequencing of a yeast-like fungal strain P6, a novel species of Aureobasidium spp.: insights into its taxonomy,evolution, and biotechnological potentials

Purpose

This study aimed to look insights into taxonomy, evolution, and biotechnological potentials of a yeast-like fungal strain P6 isolated from a mangrove ecosystem.

Methods

The genome sequencing for the yeast-like fungal strain P6 was conducted on a Hiseq sequencing platform, and the genomic characteristics and annotations were analyzed. The central metabolism and gluconate biosynthesis pathway were studied through the genome sequence data by using the GO, KOG, and KEGG databases. The secondary metabolite potentials were also evaluated.

Results

The whole genome size of the P6 strain was 25.41Mb and the G + C content of its genome was 50.69%. Totally, 6098 protein-coding genes and 264 non-coding RNA genes were predicted. The annotation results showed that the yeast-like fungal strain P6 had complete metabolic pathways of TCA cycle, EMP pathway, pentose phosphate pathway, glyoxylic acid cycle, and other central metabolic pathways. Furthermore, the inulinase activity associated with β-fructofuranosidase and high glucose oxidase activity in this strain have been demonstrated. It was found that this yeast-like fungal strain was located at root of most species of Aureobasidium spp. and at a separate cluster of all the phylogenetic trees. The P6 strain was predicted to contain three NRPS gene clusters, five type-I PKS gene clusters, and one type-I NRPS/PKS gene cluster via analysis at the antiSMASH Website. It may synthesize epichloenin A, fusaric acid, elsinochromes, and fusaridione A.

Conclusions

Based on its unique DNA sequence, taxonomic position in the phylogenetic tree and evolutional position, the yeast-like fungal strain P6 was identified as a novel species Aureobasidium hainanensis sp. nov. P6 isolate and had highly potential applications.

     

A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

Linking small-scale topography with microclimate,plant species diversity and intra-specific trait variation in an alpine landscape

Background: Small-scale topographic complexity is a characteristic feature of alpine landscapes, with important effects on alpine plant distribution.

Aims: We investigated the links between small-scale topographic complexity and resultant microclimatic heterogeneity, vascular-plant species richness and beta diversity, and realised niche width and trait variation of some target species.

Methods: We recorded temperature and soil moisture within 10 sites (40 m × 40 m) of differing topographic complexity in alpine terrain at Finse, Norway (N 60° 36?, E 7° 33?). Plant species occurrence and traits of target species were recorded in 16 sample plots at each site.

Results: Sites differed significantly in microclimatic heterogeneity, and topographically rough sites were always more heterogeneous than flatter ones. Greater species richness and turnover was associated with greater microclimatic heterogeneity, and rough sites contained 15–55% more species than flatter ones. Plant species had on average wider realised niches when growing at rough sites. Individuals of Bistorta vivipara, but not those of Luzula spicata, tended to exhibit greater phenotypic variation at rough sites.

Conclusions: Rough alpine terrains create small-scale variation in microclimate, promoting species richness and beta diversity. In the event of climate change, small-scale microclimatic heterogeneity might allow plant species to escape from regional climate change by short-distance migration to local micro-refugia. This study suggests that the opportunity for such responses would be greater in topographically complex terrains.     

Associations between 60 SNPs identified by APEX microarray and growth rate,meatiness and selection index in boars

A total of 312 boars (201 Landrace and 111 Large White) were genotyped with a custom-made low throughput genotyping microarray (called SNiPORK) based on array primer extension (APEX) technology. The results were used to association studies between genotyped SNP markers and daily gains, meat content and selection index. Among the 60 SNP markers analyzed, 14 of them showed statistically significant associations between the genotype and the level of at least one trait. In order to find extremely beneficial or unfavorable intergenic diplotype combinations, 5 SNP markers were selected: CAST A499C, MYF6 T255C, PKLR C384T, SFRS1 C1146T and TNNT3 T153C, which showed statistically significant associations at P ≤ 0.01 within one of the traits and the frequency of homozygotes with a minor allele of at least 0.1. Among 10 possible permutations, statistically significant associations were found only for a combination of SNP markers in TNNT3 × SFRS1 genes and, interestingly, for all combinations with SNP located within the calpastatin (CAST) gene commonly known as a gene influencing pork quality traits. This study also found that CAST allele A (which is beneficial for pork tenderness) is also favorable for growth rate. This effect is clearly increased with additive alleles C of myogenic factor MYF6 and troponin T3 (TNNT3) and is decreased with each allele T from serine rich splicing factor 1 (SFRS1) gene. For meat content, the most favorable genotype of calpastatin gene was AC, the effect of which was generally increased with each C allele of MYF6 and TNNT3 and decreased with each allele T from SFRS1 and PKLR (puruvate kinase) genes, respectively. The calpastatin AC genotype seemed to be beneficial for selection index, although in combination with the CC genotype of troponin T3, calpastatin genotypes AA and CC reached higher values. In the case of the combination of genotypes TNNT3 × SFRS1, the most preferable for all analyzed traits is the CC genotype of TNNT3, especially in combination with CT or CC genotypes in the SFRS1 gene. We conclude that searching for interaction effects between candidate SNPs (even of moderate influence) may lead to interesting and valuable findings enabled better understanding and applications of SNPs in pork yield and quality improvement programmes.     

Synthesis of the Human Insulin Gene: Protein Expression,Scaling Up and Bioactivity

Abstract

Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed‐batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS‐PAGE, RP‐HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.     

Growth patterns of Mytilopsis leucophaeata,an invasive biofouling bivalve in Europe

Abstract

For the first time, growth of Mytilopsis leucophaeata, an important European fouling species, was investigated. By means of growth cages, individual shell growth of three cohorts, with, respectively, initial shell lengths of ≤5 mm, 10 mm and 15 mm, was monitored in the harbour of Antwerp, Belgium, during 2003 – 2004. M. leucophaeata followed an oscillatory growth pattern with a single summer growing period per year (May to August). Growth decreased during wintertime, but never ceased completely. M. leucophaeata has an average growth rate of <3 – 6 mm year^?1. Temperature was found to be the main environmental factor affecting growth. The von Bertalanffy growth function was used to model growth of individuals ≤5 mm, resulting in L_∞ = 16.7 mm and K = 0.56. Based on a combination of growth of all three cohorts, the hypothetical growth of an average individual mussel could be modelled over a 5-year period, resulting in a maximum length >19 mm with a growth rate of 0.41. Its longevity (more than 5 years) and the positive effect of higher water temperatures on growth, combined with its high resistance to chlorination, provides M. leucophaeata with a high potential for severe and long-lasting biofouling     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

Habent Fata Sua: The Rise and Fall of Garden Plants

Background: Rare cactus in the Americas and other species worldwide are threatened species because of their high level of habitat specialisation, narrow distribution range and continuing population decline.

Aims: To identify management units (MUs) based on genetic variability and demographic structure in order to propose assertive conservation actions for Mammillaria crucigera and to provide a model case study for other species that are under similar threats.

Methods: We genotyped through eight microsatellite loci in 171 individuals and described demographic structures in six populations of this cactus based on plots of 1 m².

Results: Across populations with a mean density of 2.6 m^?2 and a total of ~500 individuals counted, 30% of the individuals were reproductive (diameter >2 cm). The total heterozygosity was low (H_O = 0.54), but the inbreeding coefficient (F_IS = 0.29) and the allele diversity (N_A = 20) were high. Four genetic groups were distinguished, although considering the demographic structure, we propose three MUs.

Conclusions: It is critical to maintain the genetic connectivity within and among MUs, which can only be achieved through cooperation between government authorities and local habitants to halt the degradation and further destruction of the remnant populations. Searching MUs allows the identification of critical areas for conservation issues for all species whose extant populations are in a fragmented landscape.     

Molecular cloning,sequence characterization,polymorphism and association analysis of porcine ROPN1 gene

The full-length cDNA sequence of one porcine gene, ROPN1, was isolated using the rapid amplification of cDNA ends (RACE) method based on one pig EST sequence which was highly homologous to the coding sequence of human ROPN1 gene. The porcine ROPN1 gene encodes a protein of 212 amino acids which shares high homology with the rhophilin associated protein 1 (ROPN1) of eight species: gray short-tailed opossum (96%), horse (95%), cattle (94%), mouse (93%), rat (92%), chimpanzee (85%), human (85%) and rhesus monkey (85%). Phylogenetic analysis revealed that the porcine ROPN1 gene has a closer genetic relationship with the ROPN1 gene of gray short-tailed opossum. Polymorphism analysis showed that there was a T/C mutation at the position of 536 bp of mRNA and this leaded to the amino acid alteration from the Arg residue to the Cys residue. PCR-Hae III-RFLP was established to detect this T/C mutation and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of first parity (P < 0.01) and all parities (P < 0.05) in Large White sows, and also significantly associated with the litter size of all parities in Landrace sows (P < 0.01). Therefore, ROPN1 gene could be a useful candidate gene in selection for increasing litter size in pigs. These data serve as a foundation for further insight into this novel porcine gene.     

Transactions of the Botanical Society of Edinburgh

Background: Due to the dry continental climate, the mountains of eastern Ladakh are unglaciated up to 6200–6400 m, with relatively large areas of developed soils between 5600 and 6000 m covered by sparse subnival vegetation. However, there are no studies on the composition of plant assemblages from such extreme elevations, their microclimates, vertical distributions and adaptive strategies.

Aims: The subnival vegetation was described and the relationship between microclimate, species distribution and species functional traits was analysed.

Methods: In total, 481 vegetation samples from 91 permanent plots, a floristic database of Ladakh and extensive microclimate measurements were used. Measurements of 15 functional traits were made and their relationship with species distribution between 4600 and 6150 m was tested.

Results: The subnival zone was characterised by extreme diurnal temperature fluctuations, a short growing season (between 88 and 153 days) and low soil temperature during the growing season (between 2.9 °C and 5.9 °C). It hosted 67 species, mainly hemicryptophytes, and ranged from ca. 5600 m to the highest known occurrence of vascular plants in the region (6150 m). The most common plant families were Brassicaceae, Asteraceae, Poaceae, Fabaceae and Cyperaceae. Subnival specialists with narrow elevational ranges represented 42% of the flora; these species were shorter, had relatively higher water content and water-use efficiency and contained more nutrients and soluble carbohydrates than species with a wider elevational range.

Conclusions: The subnival vegetation of eastern Ladakh is dominated by generalist species with wide vertical ranges and not by high-elevation specialists. These findings, in view of the vast unglaciated areas available for range extension, suggest a relatively high resilience of the subnival flora to climate change in this region.     

A novel sheep gene,MMP7, differentially expressed in muscles from black-boned sheep and local common sheep

Differences in gene expression in muscles from Chinese black-boned sheep and local common sheep were investigated using mRNA differential display. One differentially expressed novel gene was identified through semi-quantitative RT-PCR, and the full-length cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of this gene is not homologous to any of the known sheep genes, but it contains an open reading frame encoding a protein of 416 amino acids, which has high homology with matrix metallopeptidase 7 (matrilysin, uterine) (MMP7) of 10 species: bovine (93%), rhesus monkey (75%), human (74%), pig (73%), chimpanzee (73%), dog (73%), horse (72%), mouse (66%), rat (65%), and chicken (53%). Thus the novel gene can be defined as the sheepMMP7 gene. It was finally assigned to GeneID: 100192317. The phylogenetic tree analysis revealed that the sheepMMP7 gene is closely related to the bovineMMP7. Our experiment is the first one to establish the primary foundation for further research on the sheepMMP7 gene.