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Biomedical subjects

R D Klein

Publications and source records attributed to R D Klein.

At least 73 records · Page 4Linked to original sources
PubMed1988

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis.

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs (J. D. Beggs, Nature [London] 275:104-108, 1978). Intact plasmids were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.

Blotting, Southern↗
PubMed1987

Signal sequence of human preproparathyroid hormone is inactive in yeast.

The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.

Animals↗
PubMed1987

Dietary fiber and early weaning on growth and rumen development of calves.

Holstein calves were assigned to treatments of 1) pelleted prestarter (22% protein and 12% fat) and starter containing 10% alfalfa; 2) prestarter and starter containing 20% alfalfa; 3) no prestarter and starter containing 10% alfalfa; and 4) no prestarter and starter containing 20% alfalfa. Calves assigned to treatments 1 and 2 were fed 3.64 kg milk/d for 2 wk and calves assigned to treatments 3 and 4 were fed 3.64 kg milk/d for 3 wk and 1.82 kg milk/d for wk 4. Calves on treatments 2 and 3 were heavier at 10 wk but body weights and heights were similar by 6 mo. Rumen fluid and plasma measures were similar among treatments. Bull calves were assigned to treatments 1 and 3 and necropsied at 3 or 6 wk. Dry feed intakes to 3 wk and volatile fatty acid concentrations were greater for calves on treatment 1 than those on treatment 3. Wet weights of the empty reticulorumens were greater for calves on treatment 1 than those on treatment 3. Papillary development was not affected by weaning system. Calves weaned at 17 d and fed a prestarter have earlier rumen development than calves fed no prestarter and weaned later.

Animals↗
PubMed1985

Expression of homologous homeo-box-containing genes in differentiated human teratocarcinoma cells and mouse embryos.

Human chromosome 17 contains a cluster of at least three distinct homeo box regions, Hu1, Hu2, and Hu5, within a 20 kb stretch of DNA. A mouse homeo box region, Mu1, which maps to chromosome 11, was isolated and found to contain extensive nucleotide homology with a 4 kb region encompassing the Hu1 homeo box. The conservation of these chromosomal regions between man and mouse was confirmed by nucleotide sequence analysis: approximately 380 bp of DNA are more than 90% homologous and the 61 amino acids of the homeo box domain are perfectly conserved. We found that a human teratocarcinoma cell line expresses high levels of Hu1 homeo-box-containing mRNA only after differentiation of the cells following treatment with retinoic acid. In contrast, mouse teratocarcinoma cells did not express Mu1 homeo-box-containing mRNA at any stage of differentiation, whereas the expression of such transcripts was detected in mouse embryos from 10 to 17 days of gestation.

Amino Acid Sequence↗
PubMed1982

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. I. Cloning and characterization studies.

To assess the role of neighboring DNA sequences in gene regulation, poly(dA).poly(dT) and poly(dG).poly(dC) were cloned adjacent to promoters of the lactose control region. Recombinant plasmids were constructed which were suitable for large scale purification of restriction fragments containing these promoters, 95-base pair (bp) AluI fragments containing the lack operator and promoter for the lac wild type and for the catabolite gene activating the protein-independent mutant, lac UV5, were cloned into pBR322. Homopolymers of varying lengths were inserted into the -60 region of these promoters using recombinant DNA techniques. Six of the recombinant plasmids were chosen for detailed analysis: wild type (wt); wt-AT, containing 70 bp of poly(dA).poly(dT); wt-GC, containing 23 bp of poly(dG).poly(dC); UV5; UV5-AT, containing 70 bp of poly(dA).poly(dT) and finally UV5-GC, containing 43 bp of poly(dG).poly(dC). These plasmids were characterized by restriction mapping and DNA sequencing. The effects of the DNA homopolymers on the interaction of the Escherichia coli RNA polymerase with the promoters were studied using nitrocellulose filter binding. The results show that poly(dA).poly(dT) increases the level of RNA polymerase binding, whereas poly(dG).poly(dC) has no detectable effect.

Base Sequence↗
PubMed1982

Effects of Neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. II. In vitro transcription analyses.

Transcription studies were conducted on the primary promoter (which initiates at +1) of lac wild type (wt) and UV5 that had insertions of poly(dA).poly(dT) or poly(dG).poly(dC) in their -60 region. This series of recombinant DNAs was designed to study systematically the effect of neighboring DNA sequences on the lac promoter. Investigations using linear templates showed that wild type expression was enhanced by the poly(dA).poly(dT) insertion, whereas UV5 activity was not affected by this polymer. Neither the wt nor the UV5 promoter was influenced by the presence of poly(dG).poly(dC). Quantitation of abortive transcripts from supercoiled templates showed that the influence of the polymers on promoter activity was small. Comparative studies of the primary promoter with a secondary promoter (which initiates near -22), activated by 25% glycerol, were performed.

Cloning, Molecular↗
PubMed1982

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. III. High resolution thermal denaturation and circular dichroism studies.

High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments. The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC). A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs. This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments. The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R. D., and Wells, R. D. (1982) J. Biol. Chem. 257, 12962-12969). Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C. CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter. The presence of homopolymers had a marked effect on the CD spectra of the fragments.

Circular Dichroism↗
PubMed1981

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments.

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.

Chemical Phenomena↗
PubMed1979

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.

DNA Restriction Enzymes↗
PubMed1977

Scanning electron microscopical examination of gonococcal urethral exudate.

The relationship of gonococci and phagocytes in gonococcal urethral exudate was examined using a scanning electron microscope. Several stages in the phagocytic process were evident. These included pseudopodias, microvilli, and simple attachment of gonococci to phagocytic surfaces. Epithelial cells, also a part of the exudate, were examined in this study. Parallels are drawn between this and other phagocytic systems. The technique provides excellent preservation of phagocyte-bacterial interrelationships and offers a means of investigating the nature of the resistance of Neisseria gonorrhoeae to phagocytosis.

Exudates and Transudates↗
PubMed1976

Evaluation of double violet agar in the isolation of Klebsiella pneumoniae from river water.

A field evaluation of double violet agar for the isolation and presumptive identification of Klebsiella pneumoniae from water has been performed. Water from the North Oconee River, Clarke County, Ga., was cultured for presence of klebsiellae using the membrane filter technique. Colonies were presumptively identified as K. pneumoniae in the basis of their appearance on double violet agar. Such identifications were evaluated using appropriate biochemical tests. Once investigators have become familiar with cultural reactions on the medium, double violet agar can be used to indicate presence of K. pneumoniae in water with greater than 80% accuracy.

Agar↗
PubMed1976

Isolation of Klebsiella pneumoniae from lake water.

The question of the importance of aquatic borne Klebsiella pneumoniae to public health has been argued as the organism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium, double violet agar, which also differentiates K. pneumoniae from other organisms found in aquatic systems, we sampled water from three ponds and a large man-made lake, over a 3-month period. Results indicate that K. pneumoniae can be isolated consistently and in high numbers from eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleaner waters are erratic. We conclude that mere isolation of the organism as an occasional contaminant probably does not present a hazard to public health.

Escherichia coli↗