Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Correlation of ghrelin concentration and ghrelin,ghrelin-O-acetyltransferase (GOAT) and growth hormone secretagogue receptor 1a mRNAs expression in the proventriculus and brain of the growing chicken

To determine mechanisms for age-related decrease of GHS-R1a expression in the chicken proventriculus, changes in mRNA expression of ghrelin and ghrelin-O-acetyltransferase (GOAT) as well as ghrelin concentrations in the proventriculus and plasma were examined in growing chickens. Changes in expression levels of ghrelin, GOAT and GHS-R1a mRNAs were also examined in different brain regions (pituitary, hypothalamus, thalamus, cerebellum, cerebral cortex, olfactory bulb, midbrain and medulla oblongata). Ghrelin concentrations in the proventriculus and plasma increased with aging and reached plateaus at 30–50 days after hatching. High level of ghrelin mRNA decreased at 3 days after hatching, and it became stable at half of the initial level. Expression levels of GHS-R1a and GOAT decreased 3 or 5 days after hatching and became stable at low levels. Significant negative correlations were found between plasma ghrelin and mRNA levels of GOAT and GHS-R1a. Expression levels of ghrelin mRNA were different in the brain regions, but a significant change was not seen with aging. GHS-R1a expression was detected in all brain regions, and age-dependent changes were observed in the pituitary and cerebellum. Different from the proventriculus, the expression of GOAT in the brain increased or did not change with aging. These results suggest that decreased GHS-R1a and GOAT mRNA expression in the proventriculus is due to endogenous ghrelin-induced down-regulation. Expression levels of ghrelin, GOAT and GHS-R1a in the brain were independently regulated from that in the proventriculus, and age-related and region-dependent regulation pattern suggests a local effect of ghrelin system in chicken brain.     

Association of single nucleotide polymorphisms in the microRNA miR‐1596 locus with residual feed intake in chickens

MicroRNAs are an abundant class of small non‐coding RNAs that regulate gene expression. Genetic variations in microRNA sequences may be associated with phenotype differences by influencing the expression of microRNAs and/or their targets. This study identified two single nucleotide polymorphisms (SNPs) in the genomic region of the microRNA miR‐1596 locus of chicken. Of the two SNPs, one was 95 bp upstream of miR‐1596 (g.5678784A>T) and the other was in the middle of the sequence producing the mature microRNA gga‐miR‐1596‐3p (g.5678944A>G). Genotypic distribution of the two SNPs had large differences among 12 chicken breeds (lines), especially between the fast‐growing commercial lines and the slow‐growing Chinese indigenous breeds for the g.5678784A>T SNP. Only the g.5678784A>T SNP was significantly associated with residual feed intake (RFI) in the F₂ population derived from a fast‐growing and a slow‐growing broiler as well as in the pure Huiyang bearded chicken. The birds with the AA genotype of the g.5678784A>T SNP had lower RFI and higher expression of the mature gga‐miR‐1596‐3p microRNA of miR‐1596 than did those with the other genotypes of the same SNP. We also found that the expression of the mature gga‐miR‐1596‐3p microRNA of miR‐1596 was significantly associated with RFI. These findings suggest that miR‐1596 can become a candidate gene related to RFI, and its genetic variation may contribute to changes in RFI by altering expression levels of the mature gga‐miR‐1596‐3p microRNA in chicken.     

Functional analysis of an auxin-inducible DNA-binding protein gene

Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance.