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Biomedical subjects

R Korn

Publications and source records attributed to R Korn.

At least 19 recordsLinked to original sources
PubMed1992

Enhancer trap integrations in mouse embryonic stem cells give rise to staining patterns in chimaeric embryos with a high frequency and detect endogenous genes.

We have generated mouse embryonic stem cell lines that carry lacZ enhancer trap constructs integrated in their genome. Fifty-nine cell lines were analysed for lacZ expression in undifferentiated stem cells and at day 7.5, 8.5 and 12.5 of development in chimaeric embryos obtained after blastocyst injection. In 13 cell lines the lacZ reporter gene was expressed in undifferentiated stem cells ('blue', lines) as monitored by beta-galactosidase activity; 46 cell lines did not show detectable beta-galactosidase activity ('white', lines). In chimaeric embryos one-third of the analysed 59 embryonic stem cell lines gave rise to a variety of patterns. Six out of the 13 'blue' lines and 14 out of the 46 'white' lines showed spatially and temporally regulated patterns of beta-galactosidase expression and were additionally analysed on day 9.5. The majority of patterns showed staining exclusively or predominantly in structures of the developing nervous system, three patterns were observed only or predominantly in non-neuronal structures and five patterns were found exclusively in extraembryonic tissues. The analysis of DNA from cell lines that gave rise to staining patterns in chimaeric embryos showed that in 11 out of 15 cases simple integrations had occurred at a single site while in the remaining four cell lines multiple copies had integrated either at a single or at multiple sites. Flanking sequences from five reporter gene integrations have been cloned. At present, three integration sites have been analysed further and in all three cases we have identified transcribed sequences in the flanking DNA and isolated corresponding cDNA clones. The expression patterns of two of these genes were analysed by RNA in situ hybridisation. In both cases, expression of the endogenous genes was more widespread than the corresponding beta-galactosidase staining, suggesting that the reporter gene responded to only a subset of the regulatory elements of the endogenous gene. Our results demonstrate that enhancer trap integrations in embryonic stem cells can be used to efficiently identify transcriptional activation patterns during mouse embryogenesis and to isolate endogenous genes expressed in spatially and temporally regulated patterns.

Animals↗
PubMed1986

Adenovirus DNA synthesis in vitro is inhibited by the virus-coded major core protein.

The effects of the adenovirus type 2 (Ad2) structural proteins on Ad DNA synthesis in vitro have been examined. Both of the viral core proteins, polypeptides V and VII were shown to inhibit Ad2 DNA synthesis in vitro; however, only the major core protein, polypeptide VII, inhibited DNA synthesis at a ratio of protein to DNA proportional to the number of polypeptide VII molecules associated with the Ad2 DNA in the mature virion. In addition, fractions containing the precursor to polypeptide VII, pVII, were capable of inhibiting Ad2 DNA replication in vitro to the same extent as polypeptide VII. Purified polypeptide VII bound to double-stranded DNA with no apparent sequence specificity. In addition, polypeptide VII protected Ad2 DNA from digestion with micrococcal nuclease. The binding of polypeptide VII was probably responsible for the inhibition of Ad2 DNA synthesis in vitro by virtue of rendering the DNA inaccessible to viral replication proteins. These results suggest that the core proteins must be removed from the Ad2 genome before the template can function in genome replication and that assembly of pVII on Ad2 DNA can terminate the replication process.

Adenoviridae↗
PubMed1986

A major internal initiation site for the in vitro translation of the adenovirus DNA polymerase.

An open reading frame which encodes at least 90% of the adenovirus type 2 DNA polymerase gene was cloned behind the SP6 promoter and transcribed in vitro using the SP6 RNA polymerase. The resultant RNA was translated in a rabbit reticulocyte cell free system. In addition to the translation of a 120-kDa protein corresponding to the size of the complete open reading frame, the synthesis of a 62-kDa polypeptide was demonstrated. Data is presented to show that the synthesis of the 62-kDa polypeptide resulted from internal initiation of translation in frame in the middle of the message at the 11th or 12th AUG. Capping of the mRNA resulted in an increase in synthesis of the 120-kDa protein and a concordant decrease of the internally initiated polypeptide. We propose that there may be competition between the binding of the translational preinitiation complex at or near the 5' end of the mRNA and at the internal initiation site. Because of inhibition of synthesis of the 120-kDa but not the 62-kDa polypeptide by hybrid arrested translation using DNA complementary to approximately one third of the 5' Ad Pol mRNA sequences, scanning of the ribosome from the 5' end of the mRNA to the internal initiation site seemed unlikely. The sequence proximal to the 12th AUG is ACCCACCCCAUG which is similar to a noncontinuous sequence 5'AUCCACC(X)nAUG complementary to the 3' end of the 18 S rRNA. This sequence is a favored ribosome binding site based on the observation that it is the most commonly observed one at or near the 5' end of 162 mRNA's analyzed (D. R. Sargan, S. P. Gregory, and P. H. W. Butterworth, 1982, FEBS Lett. 147, 133-136).

Adenoviruses, Human↗
PubMed1986

Transgenesis by means of blastocyst-derived embryonic stem cell lines.

This study demonstrates that blastocyst-derived embryonic stem cells (ES cells) can be used as a vehicle for transgenesis. The method is nearly as efficient as other methods, and the introduced neomycin phosphotransferase (neo) gene is stably transmitted through several generations with no apparent loss in G418 resistance. An important factor contributing to the efficiency of this process is the rigorous selection, before blastocyst injection, of genetically transformed cells for in vitro developmental pluripotency. One of the advantages of the ES cell route to transgenesis is that it provides investigators with the opportunity to screen for the desired genetic alterations before reintroducing the ES cells into the animal.

Animals↗
PubMed1985

The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome.

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.

Adenoviridae↗
PubMed1984

Psychosocial adaptation of children and their parents to hospital and home hemodialysis.

Psychosocial adaptation of 20 children and adolescents treated by regular hemodialysis and their parents was analyzed by detailed semistructured interviews and questionnaires. The results in 10 patients treated in the centre and 10 followed at home were compared. The burdens of patients and parents induced by therapy as well as compliance, educational aspects and school activity in the treated children are described. Home dialysis usually provoked more fears of complications and aggressive feelings in patients and stress in parents but was superior to centre treated patients regarding social contacts and school activity of patients. From the data obtained, a comprehensive programme of psychosocial care for children with end-stage kidney disease was derived, including detailed instruction for parents and teachers of children on regular dialysis treatment.

Adaptation, Psychological↗
PubMed1983

Specific in vitro binding of a plasmid to a membrane fraction of Bacillus subtilis.

A model system has been developed to study the in vitro binding of a plasmid to the membrane fraction from Bacillus subtilis. The plasmid DNA molecule used in these studies was pSL103 (8.0 kilobases), a chimeric plasmid consisting of a Staphylococcus aureus plasmid (pUB110, 4.5 kilobases) and a DNA fragment (3.5 kilobases) from Bacillus pumilus carrying trpC+ gene. This plasmid replicates in B. subtilis cells, and its in vivo membrane binding (as well as its replication) is dependent on the product of a DNA initiation gene, dna-1, of B. subtilis. In this paper we demonstrate the in vitro specific binding of exogenous pSL103 to the isolated membrane fraction. This in vitro binding is specific to the origin-containing portion (pUB110) of pSL103. The trpC+-carrying portion neither binds to the membrane fraction nor competes with pSL103 for binding to the membrane fraction in vitro. ColE1 plasmid, which does not replicate in B. subtilis, neither binds to the B. subtilis membrane fraction nor competes with pSL103 for binding.

Bacillus subtilis↗
PubMed1978

Human spinal fluid methadone levels.

Cerebral spinal fluid (CSF) levels of methadone were measured in nine methadone maintenance patients requiring lumbar punctures for medical or surgical treatment. Concurrent serum methadone levels were also determined. The CSF concentration of methadone in all cases was a fraction of the corresponding serum level--ranging from 2 to 73%. The CSF concentrations of methadone ranged from 0.010 to 0.097 ng%. Peak methadone levels in CSF appeared approximately 3 - 8 hours after methadone administration.

Humans↗