The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide …

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total …

The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long …

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

Roseate spoonbills are colonial nesting birds with breeding grounds extending from the United States Gulf coast to the pampas of Argentina. The U.S. population suffered a severe bottleneck from 1890 to 1920. The population's recovery was slow and partially credited to migrations from Mexican rookeries, but a gene pool reduction would be expected. Five polymorphic Spoonbill autosomal short tandem repeat (STR) loci [three (GAT)n, one (AAAG)n and one (GT)n] and one Z/W-linked microsatellite exhibiting sex-specific dimorphism were isolated and characterized. The Z/W-linked STR locus accurately confirmed the sex of each bird. Allelic profiles for 51 spoonbills obtained from Dallas (Texas), Fort Worth (Texas) and Sedgwick County (Kansas) zoos revealed a non-continuous distribution of allele frequencies, consistent with the effects of a population bottleneck. Allelic frequencies also differed significantly between the isolated zoo populations. Although extra-pair copulations were suspected and difficult to document, zoos commonly used observational studies of mating pairs to determine familial relationships among adults and offspring. STR parentage analysis of recorded family relationships excluded one or both parents in 10/25 cases studied and it was further possible to identify alternative likely parents in each case. Mistaken familial relationships quickly lead to the loss of genetic variability in captive …

Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all …

The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.

Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP …