Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Sequence diversity of the intergenic spacer region of the rRNA gene of Cryptococcus albidus isolated from the skin of patients with atopic dermatitis and healthy individuals

The yeast species Cryptococcus albidus var. albidus was found to more often colonize the skin surface of patients with atopic dermatitis (77.0%, 47/61) than that of  healthy subjects (37.5%, 15/40). The intergenic spacer 1 region of the rRNA gene of this species consists of four sequence types: I, II, III and IV. Types I and II were predominant among healthy subjects and atopic dermatitis patients, respectively.     

Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase.

The nucleotide sequence of a 2301 base pair region of Saccharomyces cerevisiae DNA containing the PHR1 gene is reported. Within this region a single open reading frame of 1695 base pairs was found; using the insertional inactivation technique it was shown that part or all of this open reading frame specifies the PHR1-encoded photolyase. The amino acid sequence of the 565 amino acid long polypeptide predicted from the PHR1 nucleotide sequence was compared to the amino acid sequence of E. coli photolyase. Overall the sequence homology was 36.5%; however, two short regions near the amino terminus as well as the carboxy-terminal 150 amino acids display significantly greater sequence homology. The presence of these strongly conserved regions suggests that the yeast and E. coli photolyase possess common structural and functional domains involved in substrate and/or chromophore binding.     

Sequence diversity of yeast 2 microns RAF gene and its co-evolution with STB and REP1.

Despite the extensive study of yeast 2 microns plasmid, the exact function of plasmid-encoded RAF gene is not clear. Variants of 2 microns plasmids from industrial Saccharomyces cerevisiae yeasts were isolated and characterized. Sequencing of RAF alleles revealed about 8% nucleotide and 10% amino acid diversities between 2 microns variants of closely related strains, RAF sequence variations were correlated with STB-REP1 sequence diversity. We also used restriction fragment length polymorphism linkage to screen a large number of yeast strains from different fermentation industries. The results clearly show a tight linkage of STB-REP1-RAF variations. Thus, our observations suggest that plasmid-borne cis- and trans-acting elements co-evolved to form an optimal molecular parasite and that RAF may play a role in active plasmid partitioning.     

Sequence and expression of the chicken calcitonin gene

The avian calcitonin gene was isolated and sequenced; two mRNAs are expressed by tissue-specific alternate splicing. The peptides encoded by the mRNAs are the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Calcitonin is expressed predominantly in ultimobranchial bodies and CGRP in brain.     

Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster.

Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters.     

Convergent evolution of crystallin gene regulation in squid and chicken: The AP-1/ARE connection

Previous experiments have shown that the minimal promoters required for function of the squid SL20-1 and SL11 crystallin genes in transfected rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resembles the PL-1 and PL-2 elements of the chicken B 1-cry stallin promoter which are essential for promoter function in transfected primary chicken lens epithelial cells. Here we demonstrate by site-directed mutagenesis that the AP-1/ARE sequence is essential for activity of the squid SL20-1 and SL11 promoters in transfected embryonic chicken lens cells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase protection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/ARE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with the PL-1 element of the chicken B1-crystallin promoter. This complex formed with nuclear extracts from the lens, heart, brain, and skeletal muscle of embryonic chickens and was eliminated by competition with a consensus AP-1 sequence. The nonfunctional mutant AP-1/ ARE sequences did not compete for complex formation. These data raise the intriguing possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar cis-acting regulatory element (AP-1/ARE) for high expression in the lens. Correspondence to: S. I. Tomarev     

Sequence of the V. parahaemolyticus gene for cytoplasmic N, N'-diacetylchitobiase and homology with related enzymes.

The nucleotide sequence of the gene encoding the cytoplasmic N, N'-diacetylchitobiase [EC 3.2.1.14] from Vibrio parahaemolyticus (ATCC #27969) has been determined. The deduced peptide sequence of this unusual beta-hexosaminidase surprisingly shows minimum evolutionary relationship to two other reported N, N'-diacetylchitobiases from vibrios, except in highly conserved regions which are also homologous to lysosomal beta-hexosaminidases from eukaryotes including humans. In contrast, the two other beta-hexosaminidases from vibrios with reported sequences are much more closely related to each other. This novel 85 kDa cytoplasmic glycosyl hydrolase with restricted specificity participates in the high level utilization of chitin-derived 2-deoxy-2-acetamido-D-glucose (GlcNAc) by vibrios as one of two parallel pathways for the metabolism of N,N'-diacetylchitobiose [Bassler, B.L., Yu, C., Lee, Y.C., and Roseman, S. (1991) J. Biol. Chem. 266, 24276-24286]. These pathways use chitin-binding proteins for the adherence of the bacterial chitinase to the substrate, and an extracellular chitinase and a periplasmic chitodextrinase to produce N,N'-diacetylchitobiose. The V. parahaemolyticus cytoplasmic N,N'-diacetyl-chitobiase reported herein appears to be a unique protein, lacking a signal sequence, and genetically distant from other known chitinoclastic beta-N,N'-diacetyl-hexosaminidases. This is consistent with its limited substrate specificity to small GlcNAc terminated oligosaccharides and its cytoplasmic rather than periplasmic localization.     

Sequence features and evolutionary mechanisms in the chicken avidin gene family.

The chicken avidin gene family comprises the avidin gene (avd) and several homologous avidin-related genes (avrs). The sequences of the avr genes are nearly identical to each other but exhibit nonrandomly distributed, frequently nonsynonymous nucleotide substitutions compared to avd. In this study, we determined the genetic distances and the phylogeny of the avd and avr genes and found differences between different exons and introns. Our results suggest the involvement of biased gene conversion in the evolution of the genes. Furthermore, one of the genes was identified as a putative fusion gene. The occurrence of both gene conversion and recombination supports the models suggesting a common initiation mechanism for conversion and crossing-over. The existence of avidin-related proteins (AVRs) is currently unknown, but the putative AVRs are expected to bind biotin similarly to avidin. However, the observed sequence differences may affect the stability and glycosylation patterns of the putative AVR proteins.     

Analysis of intergenic spacer regions in the nuclear rDNA of Pharbitis nil.

The nucleotide sequence of a 4.2-kb EcoRI fragment from the intergenic region between the genes for 25S and 18S ribosomal RNA of Pharbitis nil Choisy was determined. The region contained a unique repetitive family of DNA sequences, called the RsaI family, composed of 32-bp units. The 32-bp unit was tandemly repeated in the intergenic region, and four subfamilies of repeating units were clustered as discrete blocks. The RsaI family of repeats was shown to be specific to the genus Pharbitis by Southern blot hybridization.     

Polymorphism of chicken myocyte-specific enhancer-binding factor 2A gene and its association with chicken carcass traits

Myocyte-specific enhancer-binding factor 2A (MEF2A) gene is a member of the myocyte-specific enhancer-binding factor 2 (MEF2) protein family which involved in vertebrate skeletal muscle development and differentiation. The aim of the current study is to investigate the potential associations between MEF2A gene SNPs (single nucleotide polymorphisms) and the carcass traits in 471 chicken samples from four populations. Three new SNPs (T46023C, A72626G, and T89232G) were detected in the chicken MEF2A gene. The T46023C genotypes were associated with live body weight (BW), carcass weight (CW), eviscerated weight, semi-eviscerated weight (SEW), and leg muscle weight (LMW) (P < 0.05); the A72626G genotypes were associated with BW, CW, LMW (P < 0.01) and breast muscle weight (BMW), leg muscle percentage (LMP) (P < 0.05); whereas the T89232G genotypes were associated with carcass percentage (CP) and semi-eviscerated percentage (SEP) (P < 0.05). The haplotypes constructed on the three SNPs were associated with BW, CW, LMW (P < 0.01), SEW, BMW, CP (P < 0.05). Significantly and suggestive dominant effects of diplotype H1H2 were observed for BW, CW, SEW, BMW and CP, whereas diplotype H5H5 had a negative effect on BW, CW, SEW, BMW and LMW. Our results suggest that the MEF2A gene may be a potential marker affecting the muscle trait of chickens.     

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.