Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss).

In the present study, steelhead sperm were activated in artificial tap water, ovarian fluid, activating saline, or in combinations of these media, and motility characteristics were determined using computer-assisted sperm analysis. Motility characteristics of individual sperm were then assessed to test the hypothesis that motile sperm are distributed among discrete subpopulations and that their distribution is influenced by the activation medium. Analysis with k-means clustering detected three discrete motile sperm subpopulations in steelhead semen, regardless of the activation medium. Based on multivariate analysis of variance, proportions of these subpopulations did not differ between sperm activated with ovarian fluid and activating saline, or any combination of these two media. However, subpopulation distributions for sperm activated with either ovarian fluid or activating saline were influenced by the level of dilution of these media in artificial tap water. There was an increase in the number of sperm in high velocity (curvilinear), high straightness, and high wobble subpopulation with increased levels of ovarian fluid or activating saline. The change in sperm motility characteristics with a change in activation medium may play a role in normal fertilization, as discharged sperm pass from seminal plasma and water through ovarian fluid en route to the egg.

Recombination is suppressed over a large region of the rainbow trout Y chromosome.

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy-163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.

Conservation genetics of the fisher (Martes pennanti) based on mitochondrial DNA sequencing.

Translocation of animals to re-establish extirpated populations or to maintain declining ones has often been carried out without genetic information on source or target populations, or adequate consideration of the potential effects of mixing genetic stocks. We consider the conservation status of the fisher (Martes pennanti) and evaluate the potential genetic consequences of past and future translocations on this medium-sized carnivore by examining population variation in mitochondrial control-region sequences. We sampled populations throughout the fisher's range in North America including five populations unaffected by translocations and two western populations that had received long-distance translocations. Twelve haplotypes showed little sequence divergence. Haplotype frequencies differed significantly among subspecies and between populations within subspecies. Analysis of molecular variance (amova) and neighbour-joining analyses of haplotype relationships revealed population subdivision similar to current subspecies designations, but which may reflect an isolation-by-distance pattern. Populations in Oregon and in Montana and Idaho received several translocations and each showed greater similarity to the populations where translocations originated than to adjacent populations. Additional sequences obtained from museum specimens collected prior to any translocations suggest historical gene flow among populations in British Columbia, Washington, Oregon, and California. Anthropogenic impacts in that region have greatly reduced and isolated extant populations in Oregon and California. Future translocations may be necessary to recover populations in Washington and portions of Oregon and California; our results indicate that British Columbia would be the most appropriate source population.

Steric hindrance regulation of the Pseudomonas aeruginosa amidase operon.

Expression of the amidase operon of Pseudomonas aeruginosa is controlled by AmiC, the ligand sensor and negative regulator, and AmiR the transcription antitermination factor activator. We have titrated out AmiC repression activity in vivo by increased AmiR production in trans and shown AmiC regulation of the antitermination activity of AmiR by a steric hindrance mechanism. In the presence of the co-repressor butyramide we have isolated a stable AmiC.AmiR complex. Addition of the inducing ligand acetamide to the complex trips the molecular switch, causing complex dissociation and release of AmiR. The AmiC.AmiR butyramide complex exhibits acetamide-dependent, sequence-specific RNA binding activity and a K(d) of 1.0 nm has been calculated for the AmiR.RNA interaction. The results show that amidase operon expression is controlled by a novel type of signal transduction system in which activity of a site-specific RNA binding activator is regulated via a sequestration mechanism.

Structural adaptation to selective pressure for altered ligand specificity in the Pseudomonas aeruginosa amide receptor, amiC.

The AmiC protein in Pseudomonas aeruginosa is the negative regulator and ligand receptor for an amide-inducible aliphatic amidase operon. In the wild-type PAC1 strain, amidase expression is induced by acetamide or lactamide, but not by butyramide. A mutant strain of P. aeruginosa, PAC181, was selected for its sensitivity to induction by butyramide. The molecular basis for the butyramide inducible phenotype of P.aeruginosa PAC181 has now been determined, and results from a Thr-->Asn mutation at position 106 in PAC181-AmiC. In the wild-type PAC1-AmiC protein this residue forms part of the side wall of the amide-binding pocket but does not interact with the acetamide ligand directly. In the crystal structure of PAC181-AmiC complexed with butyramide, the Thr-->Asn mutation increases the size of the ligand binding site such that the mutant protein is able to close into its 'on' configuration even in the presence of butyramide. Although the mutation allows butyramide to be recognized as an inducer of amidase expression, the mutation is structurally sub-optimal, and produces a significant decrease in the stability of the mutant protein.

Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex.

Inducible expression of the aliphatic amidase operon in Pseudomonas aeruginosa is controlled by an antitermination mechanism which allows production of the full-length transcript only in the presence of small-molecule inducers, such as acetamide. Ligand-regulated antitermination is provided by AmiC, the ligand-sensitive negative regulator, and AmiR, the RNA-binding positive regulator. Under non-inducing or repressing growth conditions, AmiC and AmiR form a complex in which the activity of AmiR is silenced. The crystal structure of the AmiC-AmiR complex identifies AmiR as a new and highly unusual member of the response-regulator family of bacterial signal transduction proteins, regulated by sequestration rather than phosphorylation. Comparison with the structure of free AmiC reveals the subtle mechanism of ligand-induced release of AmiR.

Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon.

In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon. A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination. Site-directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5' and 3' ends of the proposed AmiR binding site which are independently essential for antitermination. A T7 promoter/RNA polymerase-driven system shows AmiR-mediated antitermination, demonstrating a lack of promoter/polymerase specificity. Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC-AmiR complex has been identified and isolated. Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC. AmiC-AmiR-anti-inducer is a stable dimer-dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination.

Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS).

The nucleotide sequence of the amidase operon of Pseudomonas aeruginosa has been completed and two new genes identified amiB and amiS. The complete gene order for the operon is thus amiEBCRS. The amiB gene encodes a 42-kDa protein containing an ATP binding motif that shares extensive homology with the Clp family of proteins and also to an open reading frame adjacent to the amidase gene from Rhodococcus erythropolis. Deletion of the amiB gene has no apparent effect on inducible amidase expression and it is thus unlikely to encode a regulatory protein. A maltose-binding protein-AmiB fusion has been purified and shown to have an intrinsic ATPase activity (Km = 174 +/- 15 mM; Vmax = 2.4 +/- 0.1 mM/min/mg), which is effectively inhibited by ammonium vanadate and ADP. The amiS gene encodes an 18-kDa protein with a high content of hydrophobic residues. Hydropathy analysis suggests the presence of six transmembrane helices in this protein. The AmiS sequences is homologous to an open reading frame identified adjacent to the amidase gene from Mycobacterium smegmatis and to the ureI gene from the urease operon of Helicobacter pylori. AmiS and its homologs appear to be a novel family of integral membrane proteins. Together AmiB and AmiS resemble two components of an ABC transporter system.

Transcriptional analysis of the amidase operon from Pseudomonas aeruginosa.

The transcriptional start point for the amidase structural gene (amiE) of Pseudomonas aeruginosa has been identified, and the promoter (pE) has been shown to function constitutively, as predicted for a system regulated by transcription antitermination. Northern (RNA) analysis results show that in cells grown under inducing conditions, a major 1.3-kb amiE transcript arises from pE, and in addition, a larger transcript of approximately 5.0 kb in length has been shown to derive from the same promoter, encoding all of the genes of the operon. DNA sequencing and S1 nuclease mapping have located a transcription terminator downstream of amiE, which terminates approximately half of the pE transcripts. Previously, two RpoN-dependent promoter-like sequences (pN1 and pN2) were identified upstream of the negative regulator gene, amiC, and we have now constructed a promoter probe vector which shows weak constitutive promoter activity within this region. This promoter would be expected to provide basal levels of expression of the amiC and amiR regulatory genes to allow induction of the system.

Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein.

Amide-inducible expression of the aliphatic amidase system of Pseudomonas aeruginosa can be reconstituted in Escherichia coli with only the amidase structural gene amiE, the negative regulator amiC and the positive regulator amiR, a transcription antitermination factor. Complementation experiments in E. coli suggest that negative control of amidase expression by AmiC is mediated by a protein-protein interaction with AmiR. Purified AmiC binds acetamide with a KD of 3.7 microM in equilibrium dialysis studies, and therefore AmiC appears to be the sensory partner of the AmiC/AmiR pair of regulatory proteins, responding to the presence of amides. Sequence analysis techniques suggest that AmiC is a member of the structural family of periplasmic binding proteins, but has a distinct and novel cytoplasmic role.

Positive control of gene expression in procaryotes.

In procaryotes three types of control network are involved with the regulation of gene expression: global control networks, operon/single gene specific regulation and temporal control systems. The mechanisms used for positive control include the use of new components of the transcription apparatus such as new RNA polymerases and sigma factors, the use of activator proteins and the use of transcription antitermination systems. For the second two mechanisms, secondary negative control systems may also operate to regulate the activity of activator proteins and transcription antitermination factors. Thus, only very rarely is regulation achieved by just a simple positive control system. Positive control circuits allow tight regulation of gene expression that is fail-safe, in that most mutant changes will lead to the system becoming non-inducible rather than lead to constitutive gene expression.

Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa.

The negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa has been purified from an over-expressing clone and crystalized. Crystals of diffraction quality were obtained from polyethylene glycol 4000 and ammonium sulphate. AmiC crystallizes in P4(2)2(1)2 (a = 104.4 A, c = 66.6 A) with one subunit in the asymmetric unit. Crystals diffract beyond 2.8 A.

Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa.

The nucleotide sequence of a 1001 bp ClaI/XhoI DNA fragment encoding the amidase regulator gene (amiR) from Pseudomonas aeruginosa has been determined. The sequence derives from strain PAC433, a constitutive high expressing amidase mutant, and contains two overlapping open reading frames. Analysis of the sequence has identified one of the reading frames as amiR. The gene encodes a 196 amino acid polypeptide which shows a strong bias towards codons with G or C in the third position. The amiR gene shows no sequence homology with other bacterial regulator proteins.

The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa.

The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised.

Alignment of cloned amiE gene of Pseudomonas aeruginosa with the N-terminal sequence of amidase.

A restriction enzyme map was constructed for 5.1-kb fragment of Pseudomonas aeruginosa DNA inserted into plasmid pBR322. Restriction enzyme sites were matched to the N-terminal amino acid sequence of amidase to obtain alignment of the amiE gene within the cloned fragment.

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa.

The amidase genes of Pseudomonas aeruginosa were inserted into a lambda replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant lambdaami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with lambdaami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of lambdaami were made by introducing Q-, S-, mutations. Cultures of E. coli infected with lambdaamiQ-S- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificty, thermal stability and immunological cross-reaction.