Optimal Staining and Sample Storage Time for Direct Microscopic Enumeration of Total and Active Bacteria in Soil with Two Fluorescent Dyes

Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.     

Thermodynamics of hydrophobic polyacids

The thermodynamics of deprotonating hydrolyzed 1-1 copolymers of maleic anhydride with pentyl, hexyl and octyl vinyl ether was investigated by calorimetric and potentiometric methods. These polyacids undergo a transition from a compact to a random coil conformation upon ionization in aqueous media. The results were compared with those obtained previously for similar copolymers with smaller alkyl side-chains. The contributions of the enthalpy and entropy to the free energy were analyzed. The major effects appeared to be related to the charging of the compact form of the polyacids, the electrostriction of water by the completely ionized dicarboxylate groups and the reorganization of water around newly exposed alkyl side-chains arising from the conformational transition.     

The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

cDNAs encoding members of a family of proteins related to human sterol carrier protein 2 and assignment of the gene to human chromosome 1 p21----pter.

Sterol carrier protein 2 (SCP2) is believed to play a key role in intracellular lipid movement. Here we report the cloning and nucleotide sequences of cDNAs encoding SCP2-related proteins of 58.85 kD and 30.8 kD and the assignment of the SCP2 gene to human chromosome 1 p21-pter. The SCP2-related proteins share common deduced carboxyl amino acid sequences with SCP2 and the cDNAs have a common 3' untranslated nucleotide sequence. The mRNAs encoding these proteins increased in a coordinate fashion as human placental cytotrophoblasts differentiated into syncytiotrophoblasts in culture. Our observations document the existence of a family of related proteins encoded by the human SCP2 gene.     

Similarity between the amino-terminal portion of mammalian 58-kD sterol carrier protein (SCPx) and Escherichia coli acetyl-CoA acyltransferase: evidence for a gene fusion in SCPx.

A computer analysis of the amino acid sequences of rat and human 58-kD sterol carrier protein and Escherichia coli acetyl-CoA acyltransferase reveals that the two proteins have a segment of about 350 residues with strong sequence similarity. The ALIGN comparison scores for the rat and human sterol carrier proteins and the E. coli enzyme are 8.25 and 8.8 SD, respectively. The catalytically active cysteine of E. coli acetyl-CoA acyltransferase (cysteine 91) aligns with cysteine 93 and cysteine 94 on human and rat 58-kD sterol carrier protein, respectively.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.