Rodney J. Sobieski

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT‐I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT‐III and MT‐IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT‐I and ‐II genes. MT‐III and MT‐IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT‐I and MT‐II mRNAs. In contrast, MT‐III and MT‐IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT‐I and ‐II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT‐I, MT‐III, and MT‐IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co‐expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT‐I, ‐II, and ‐III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin‐lipopolysaccharide (LPS) and Zn are powerful inducers of MT‐I and MT‐II gene expression in many adult organs, whereas these agents apparently have little effect on MT‐III and MT‐IV gene expression. Neither of these agents significantly effected levels of decidual MT‐III or MT‐IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT‐I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT‐I and MT‐III mRNA levels remained elevated while MT‐IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation.

Evolution of avian metallothionein: DNA sequence analyses of the turkey metallothionein gene and metallothionein cDNAs from pheasant and quail.

SummaryThe turkey metallothionein gene (tkMT) was isolated from a phage lambda-turkey genomic DNA library by virtue of high identity with chicken MT cDNA. The nucleotide sequences of the proximal 240 by of the 5′-flanking region, of each of the three exons, and of the intron/exon boundaries were determined. Comparisons of the nucleotide sequences of the tkMT and cMT genes revealed (1) absolute conservation of intronic DNA immediately flanking each respective intron/exon boundary, (2) high conservation (95.6%) of exonic DNA encoding translated regions of the mRNA, and (3) high conservation (95%) of exonic DNA encompassing the putative transcription start point and polyadenylation signals. Sequence comparisons of the tkMT and cMT promoters regions near the TATA box revealed that both promoters contain a highly conserved proximal metal-responsive enhancer (MRE-enhancer) motif. The deduced amino acid sequence (63 amino acids) of tkMT was identical with that of cMT. In order to further explore the degree of conservation of the protein coding regions of avian MT genes, partial MT cDNAs from turkey, quail (qMT), and pheasant (pMT) were amplified using the reverse transcriptase-polymerase chain reaction (RT-PCR) and primers corresponding to the amino- and carboxyl-terminal coding regions of cMT mRNA. RT-PCR reaction products were cloned and the DNA sequences of multiple cDNA clones from each species were determined. The results suggest the existence of a single MT mRNA in zinc-treated liver from turkey and pheasant and the existence of a major and possibly a minor MT mRNA in quail. Comparisons of the nucleotide sequences of the predominant cMT, tkMT, qMT, and pMT cDNAs revealed exceptionally high identity (97%) and the deduced MT peptide sequences (residues 15-57) were identical. The data suggest that the nucleotide sequence of these avian MT cDNAs is evolving at a predictably slow rate, but that the amino acid sequence of the encoded protein is exceptionally well conserved.

Antimicrobial susceptibility of staphylococci isolated from the faeces of wild turkeys (Meleagris gallopavo)

Aims: The purpose of this study was to investigate the staphylococcal flora associated with wild turkey populations.

Testing a non-lethal DNA isolation technique for freshwater mussels that is compatible with polymerase chain reaction (PCR) based studies

Abstract In this study, a non-lethal DNA isolation technique was developed for freshwater mussels. A total of 45 Quadrula quadrula, 21 Q. metanevra, and 19 Q. pustulosa were collected from the Neosho and Verdigris Rivers in eastern Kansas. DNA was successfully isolated from 82 mussels using the non-lethal technique developed in this study. Spectrophotometer analysis of the 82 DNA samples resulted in an average DNA concentration of 488 ng/μl. Agarose gel electrophoresis of polymerase chain reaction products obtained using the purified mussel DNA demonstrated high quality, reproducible amplification products. All mussels remained alive for several months after tissue biopsy in the laboratory. A mark and recapture study was also performed to confirm survivability, thereby ensuring our procedure was non-lethal. Recapture efforts resulted in a 56% live recovery of mussels from the Neosho River and a 78% live recovery from mussels in the Verdigris River approximately four and one-half months after their release.

Cell agglutination by a novel cell surface sialoglycopeptide inhibitor and the relationship between its protease and biological activities.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

Genotyping by Randomly Amplified Polymorphic DNA is Influenced by the Method of DNA Preparation

Abstract Randomly amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) was performed on DNA templates prepared from thirty veterinary Salmonella typhimurium isolates. The templates were isolated by four different methods, each differing in their level of purity as determined by 260 nm/280 nm ratios. Dendrogram analysis of RAPD-PCR amplification patterns resulted in the organisms being placed into a range of 26-12 discrete groupings dependent on the DNA template isolation method used. Thus, DNA purity plays an important role in amplification patterns obtained by RAPD-PCR and investigators must consider this variable before making judgements on the genetic relatedness of a group of organisms.