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Biomedical subjects

T Friedmann

Publications and source records attributed to T Friedmann.

At least 145 records · Page 8Linked to original sources
PubMed1984

Partial phenotypic correction of human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyltransferase-deficient) lymphoblasts with a transmissible retroviral vector.

A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.

Animals↗
PubMed1984

Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation.

To explore the role of DNA methylation in maintaining dosage compensation of X chromosome-linked genes and in regulating the transcriptional activity of "housekeeping" genes, we characterized DNA methylation of active, inactive, and derepressed alleles at the locus for hypoxanthine phosphoribosyltransferase (HPRT) on the human X chromosome. The methylation of Hpa II and Hha I sites in HPRT alleles on the active X chromosome was the same in all tissues. The consensus pattern includes hypomethylation of 5' clustered sites and extensive methylation of the 3' sequence. The striking feature of methylation of inactive X-chromosome alleles is nonuniformity and less extensive hypomethylation of the 5' cluster. Analysis of HPRT alleles reactivated in response to 5-azacytidine showed at least partial restoration of the consensus pattern. These observations indicate that methylation of housekeeping genes on the X chromosome is the same as that of autosomal ones and that the overall pattern and methylation of multiple sites within a cluster may cooperate to facilitate transcription. Furthermore, the fidelity of methylation of the active allele and the extensive drift in methylation of the inactive allele suggest that mechanisms involved in X-chromosome dosage compensation may be directed at the active rather than inactive X chromosome.

Alleles↗
PubMed1984

Swimming behavior of X and Y human sperm.

A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.

Cell Separation↗
PubMed1984

Structure of the heterogeneous L-S junction region of human cytomegalovirus strain AD169 DNA.

The genome of human cytomegalovirus strain AD169 contains a region of heterogeneity located at the junction between the long (L) and short (S) components of the viral DNA. Twelve cloned L-S junction fragments were studied by using the restriction enzymes HaeII and XhoI. The region of heterogeneity was localized within a single HaeII restriction fragment. The enzyme XhoI was used to subdivide this region and revealed the presence of three types of heterogeneity within the junction fragments. Each of the cloned junction fragments contained one of the following fragments: 0.553, 0.95, or 1.35 kilobase pairs (referred to as class I heterogeneity). Class II heterogeneity was defined as the presence of tandem duplications of class I fragments. In addition, a variable number (0 to 5) of a 0.2-kbp fragment (class III heterogeneity) was observed. Mapping of these fragments with partial XhoI digestions revealed that the class I and class III heterogeneous fragments were adjacent. The DNA sequence of the smallest cloned L-S junction fragment was determined and analyzed. This junction fragment contained a single 0.553-kbp XhoI fragment and no copies of the 0.2-kbp fragment. The 0.553-kbp XhoI fragment was similar in structure to the a-sequences of herpes simplex virus types 1 and 2. In addition, a region of homology was found between the a sequences of herpes simplex virus types 1 and 2 and the 0.553-kbp XhoI fragment from the human cytomegalovirus junction.

Base Sequence↗
PubMed1983

Elements in the long terminal repeat of murine retroviruses enhance stable transformation by thymidine kinase gene.

We have investigated the effects of long terminal repeats (LTRs) of murine retroviruses on the frequency of obtaining stable transfectants by the herpes virus thymidine kinase (TK) gene. The results indicate that addition of LTRs enhances the number of TK+ transformants by 10-20 fold. A 5-12 fold enhancement was also observed when chromosomal DNA from either human or hamster cells was mixed with a plasmid containing LTR sequences and transfected onto LTR- cells. The LTR sequences involved in the enhancement were localized in the region which contains tandem repeats. All other regions of the LTR did not show any enhancement of stable TK+ transfectants. The location or the orientation of the enhancer sequences with respect to the TK gene did not exert any influence on the frequency of transformation. The enhancement effect does not appear to be linked to either increased numbers of chromosomal integrations or elevated levels of transcription of the TK gene.

Animals↗
PubMed1983

A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT.

A cDNA corresponding to the human gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been ligated into murine retroviral vectors such that it is under the transcriptional control of viral long terminal repeats. Transfection of HPRT- cells followed by superinfection with various helper viruses has led to the rescue of chimeric virus capable of transmitting the HPRT+ phenotype to HPRT- rodent or human cells. These genetically transformed cells contain authentic human HPRT at levels similar to normal HPRT+ cells.

Animals↗
PubMed1983

Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase.

We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRT protein. Transfection of HPRT-deficient mouse LA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.

Amino Acid Sequence↗
PubMed1983

Study of the analgesic effects of galanthamine, a cholinesterase inhibitor.

The effects of galanthamine, an alkaloidal anticholinesterase agent whose chemical structure bears similarity to codeine, was studied in various animal and isolated organ tests. Antinociceptive activity of galanthamine given subcutaneously was detected and compared to physostigmine and morphine in the rat hot plate test. Naloxone partially blocked the effect of galanthamine but not that of physostigmine. Both cholinesterase inhibitors provided analgesia in the mouse acetic acid writhing test. They potentiated the effect of morphine in the rat hot plate test but inhibited the barbiturate anaesthesia potentiation of morphine in the rat. While galanthamine provided analgesia in the intact animal, it failed to produce opiate-like activity in such isolated organs as longitudinal muscle strip of the guinea-pig ileum, mouse vas deferens, and cat nictitating membrane.

Analgesics↗
PubMed1982

Direct evidence that eseroline possesses morphine-like effects.

The opiate-like effects of eseroline, a physostigmine derivative, were studied in different tests. The antinociceptive effect of eseroline given s.c. and intracerebrally could be detected in the rat hot plate test and was reversed by naloxone. The apparent pA2 values of naloxone demonstrated with eseroline and morphine were found to be equal, suggesting an effect on similar receptors. Eseroline also had opiate agonist activity on the isolated longitudinal muscle strip of guinea pig ileum and isolated nictitating membrane of the cat: presynaptically, it inhibited the contractions evoked by stimulation and its effect was antagonized by naloxone. Eseroline reduced acetylcholine release from the myenteric plexus of the longitudinal muscle strip when the cholinesterases had been inhibited by physostigmine. In addition, it was also found that eseroline antagonized the inhibitory effect of normorphine in the longitudinal muscle strip and potentiated the effect of exogenous acetylcholine on smooth muscle, both effects being attributed to its anticholinesterase activity. The analgesic effect of eseroline was not related to its anticholinesterase activity.

Acetylcholine↗
PubMed1982

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium. DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT. Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322. Colonies of transformed Escherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared. Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA. One of these, pBR1.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line. These results indicate that these clones represent human HPRT gene fragments. This has been confirmed by using pBR1.5 as a probe to isolate an authentic and expressible human HPRT cDNA clone from a library prepared by H. Okayama and P. Berg.

Cloning, Molecular↗
PubMed1981

Analysis of transcription of the human Alu family ubiquitous repeating element by eukaryotic RNA polymerase III.

A series of clones that contain human Alu family elements are actively transcribed in soluble in vitro RNA polymerase III systems. The 5' ends of the in vitro transcripts are located about 170 nucleotides upstream of the eponymous Alu I site of the repeat, while a region associated with specifying of the initiation site for in vitro transcription lies in the region between 79 and 106 nucleotides upstream of the central Alu site. Thus, the RNA polymerase III transcription unit defined by the human Alu family is similar to other RNA polymerase III transcription units in possessing an internal region that is required for active transcription in vitro.

Animals↗
PubMed1981

Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells.

F9 mouse embryonal carcinoma cells are resistant to productive infection by wild-type polyoma virus. Continued passage of F9 cells initially infected with wild-type polyoma virus eventually leads to the selection of polyoma virus mutants that are capable of productive infection of undifferentiated F9 cells. Three mutants, PyF101, PyF111 and PyF441, have been plaque-purified and examined. All three PyF mutant DNAs are altered from the wild-type sequence in the Pvu II-4 fragment that spans 67.6 to 70.2 map units on the polyoma genome. PyF441 has a single base change of A to G at 69.6 map units. PyF101 and PyF111 DNAs also contain this point mutation at 69.6 map units. In addition, PyF101 and PyF111 DNAs have exact tandem duplications of 54 and 31 bp, respectively, of sequences encompassing the point mutation, and both copies of the tandem duplication have the point mutation. Other than these changes, no difference exists in the nucleotide sequences of wild-type and PyF mutant DNAs from the BcI I site at 65.6 map units clockwise through the origin of viral DNA replication to the BgI I site at 72.2 map units. DNA infections of F9 cells with wild-type-mutant hybrid DNAs formed by ligation of heterologous combinations of the small and large DNA fragments generated by double digestion with the restriction enzymes BcII and BGI I show that the DNA sequence changes described above are responsible for the ability of the PyF mutants to infect F9 cells.

Animals↗
PubMed1981

Nucleotide sequences at the termini of phi 29 DNA.

The nucleotide sequences of the first 422 base pairs from the left-hand end and the first 274 base pairs from the right-hand end of phi 29 DNA were determined by using the chemical degradation method of Maxam and Gilbert. The data indicate that phi 29 DNA has inverted terminal repetitions that are six base pairs long 5' (-A-A-A-G-T-A-). No perfectly self-complementary sequence exists within the terminal regions of phi 29 DNA, suggesting that DNA replication via a self-priming mechanism is improbable. The putative early promoter sequences were found in both ends of the phi 29 DNA. The results of the sequence determination are discussed in relation ship to models proposed for the mechanism of replication of linear DNA molecules.

Base Composition↗
PubMed1981

Nucleotide sequence at polyoma VP1 mRNA splice sites.

Double-stranded DNA complementary to total cytoplasmic polyadenylated RNA isolated late in infection from polyoma virus-infected mouse 3T6 cells was cloned in Escherichia coli by using the large HindIII-BamHI fragment of pBR322 plasmid DNA. Polyoma-specific DNA inserts were detected by hybridization, and then nucleotide sequences were determined from two clones. The sequence of the (formula see text) with prototypical mammalian splice sites, and the dashed arrows indicate possible alternative splice sites leading to the same spliced product. A sequence of 897 nucleotides was spliced out of the primary transcript during the processing of the mature VP1 mRNA. Restriction enzyme mapping with four other independently isolated clones indicates that these are the major splicing signals for the VP1 message. The distal splice site is 48 nucleotides upstream from the initiator codon.

Base Sequence↗
PubMed1981

Nucleotide sequence changes in polyoma ts-a mutants: correlation with protein structure.

The mutations in three polyoma ts-a mutants have been determined. Two mutants, ts-25 and ts-52, have different single-base changes at the same position (2883) in the early region corresponding to a conserved glycine residue very near the C-terminus of the polyoma large T antigen. Mutant ts-48 has a single-base change at position 2341, as well as a second change at position 1228, in the region of large T antigen shared with medium T antigen.

Amino Acid Sequence↗