Karl Köhrer

Preparation of high molecular weight RNA

Publisher Summary This chapter presents the procedure for preparation of high molecular weight RNA. High molecular weight RNA can be isolated from haploid or diploid yeast cells as well as from ascospores. The conditions under which the yeast cells are grown (complete or selective media) do not influence the preparation procedure, although higher yields of RNA are generally obtained from cells grown in rich medium. Procedure requires some special precautions owing to the ubiquitous presence of RNA-degrading enzymes. For this reason it is important to wear gloves during the isolation and preparation procedure, and it is essential to separate all chemicals, tubes, and tips used for RNA preparation from commonly used materials. All glassware has to be baked for 2 hr at 200°; commercially available sterile plastic tubes or tips do not have to be specially pretreated, because they seem to be free of fibonucleases. Because the degradation of RNA by RNases is a time-dependent reaction, it is also advisable to work as fast as possible.

Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

Prevalence of gyrA, gyrB, parC, and parE Mutations in Clinical Isolates of Streptococcus pneumoniae with Decreased Susceptibilities to Different Fluoroquinolones and Originating from Worldwide Surveillance Studies during the 1997-1998 Respiratory Season

ABSTRACT From 8,419 worldwide clinical isolates of Streptococcus pneumoniae obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, onlyparC (Ser-79→Tyr) and gyrA (Ser-81→Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxacin/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.

Characterization of grlA,grlB, gyrA, and gyrB Mutations in 116 Unrelated Isolates of Staphylococcus aureus and Effects of Mutations on Ciprofloxacin MIC

ABSTRACT One hundred sixteen unrelated clinical isolates ofStaphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB,gyrA, and gyrB gene loci. Two mutations withingrlA (located at codons 80 and 84) and two mutations withingyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.

Lysophosphatidic acid, a novel lipid growth factor for human thyroid cells: over-expression of the high-affinity receptor edg4 in differentiated thyroid cancer.

Lysophosphatidic acid (LPA) is a small lipid mediator with pleiotropic biological activities, e.g., the regulation of cellular proliferation and various aspects of cellular physiology. Signal transduction is achieved by binding to 2 high‐affinity receptors, EDG2 and EDG4, and a group of low‐affinity receptors, EDG1–7, all belonging to the superfamily of G protein–coupled receptors. We examined the growth‐regulatory effects of LPA in primary cultures of 8 goiters and 1 papillary thyroid cancer. We further assessed mRNA expression of high‐affinity receptors EDG2 and EDG4 in 14 normal thyroids, 29 papillary thyroid cancers, 7 follicular thyroid cancers and 13 goiters by quantitative RT‐PCR. We also identified mRNA expression of phospholipase A2 and LPA acyltransferase in fresh thyroid tissues derived from various sources. At concentrations of 10, 50 and 150 μM, LPA induced a 2‐fold rise of proliferation (p < 0.001) and acted as strongly as thyrotropin. The combination of LPA and TSH produced significant synergistic effects compared with each substance alone (p < 0.05). Normal thyroid, goiter and papillary or follicular thyroid cancer expressed 2 high‐affinity cognate LPA receptors, EDG2 and EDG4. EDG4 receptor mRNA expression was increased 3‐fold in differentiated thyroid cancer (p < 0.01), both papillary (p < 0.01) and follicular (p < 0.05), compared to normal thyroid or goiter. Overall expression of EDG2 receptor was unchanged in malignancy; however, increased EDG2 expression in individual samples correlated with lymphonodular metastasis (p = 0.01). Thus, lipid mediators are a novel class of factors involved in the control of proliferation in the human thyroid. Altered mRNA expression of the high‐affinity LPA receptor EDG4 suggests a role in the pathogenesis of differentiated thyroid cancer.

The VPS4 gene is involved in protein transport out of a yeast pre-vacuolar endosome-like compartment

Abstract Four yeast mutants were isolated in a screen for dominant-negative vacuolar protein-sorting mutants, secreting a carboxypeptidase Y-invertase hybrid protein. In addition to defects in the sorting/transport of soluble vacuolar hydrolases, the mutants accumulated a pre-vacuolar endosome-like compartment. The mutant alleles causing the defects were identified as the members of the VPS4 gene locus, each harbouring single-point mutations leading to amino-acid exchanges at positions 233 (E233Q), 211 (E211 K), and 178 (G178D). These mutations all reside within a 200 amino-acid-long ATPase module, common to members of the AAA-protein family. The VPS4 gene product shows homology to the yeast Sec18p (50% similarity and 25% identity), which is involved in several vesicle-mediated protein transport steps and homotypic membrane fusion events. Disruption of the VPS4 gene leads to a recessive vacuolar protein-sorting phenotype. About 40% of newly synthesized CPY is secreted as the Golgi-modified p2CPY precursor form. Transport of secretory proteins to the plasma membrane is normal as demonstrated by the secretion of invertase and α-factor. The α-factor, however, is secreted as a partially processed precursor, caused by defects in late Golgi function. The vps4 mutants also exhibit defects in fluid-phase endocytosis, as demonstrated by the accumulation of Lucifer Yellow in a pre-vacuolar endosome-like compartment. Based on the pleiotropic phenotype of the vps4 mutants and on the sequence homology to NSF/Sec18p, we propose that the VPS4 gene product is required for efficient transport out of the pre-vacuolar endosome-like compartment.

Development of Resistance to Ciprofloxacin, Rifampin, and Mupirocin in Methicillin-Susceptible and -Resistant Staphylococcus aureus Isolates

ABSTRACT A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described forStaphylococcus aureus. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant S. aureus (MRSA). No differences in MR, DIFF, and mutations ingrlA and gyrA were detected between methicillin-susceptible S. aureus and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.

Oligonucleotide microarray analysis of differential transporter regulation in the regenerating rat liver

Abstract: Aims: The aim of this study was to investigate the regulation of hepatic transport systems during liver regeneration.

In Vitro Development of Resistance to Six Quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus

ABSTRACT Streptococcus pneumoniae, Streptococcus pyogenes, andStaphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

Increased prevalence of class I integrons in Escherichia coli, Klebsiella species, and Enterobacter species isolates over a 7-year period in a German university hospital

ABSTRACT The prevalence of integrons in five enterobacterial species was analyzed in 900 blood culture isolates from 1993, 1996, and 1999. Remarkably, the prevalence increased from 4.7% in 1993 to 9.7% in 1996 and finally to 17.4% in 1999 (P < 0.01). Within 7 years the combined percentage of P1 strong promoters and P1 weak plus P2 active promoters with high transcription efficacies has increased from 23.1 to 33.3 and finally 60% (P< 0.05).