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W Lindenmaier

Publications and source records attributed to W Lindenmaier.

15 recordsLinked to original sources
PubMed1990

Cloning and sequence analysis of the human parathyroid hormone gene region.

A region of 50 kb around the human PTH gene was cloned and mapped by restriction analysis. Sequence analysis was performed and 3270bp determined, completing the sequence of the gene. The nucleotide sequence was analysed with regard to homology between human, bovine and rat PTH genes, and various potential cis-acting regulatory elements were identified. The gene region lacks an obvious CpG island. The PTH gene region in patients suffering from (pseudo)-hypoparathyroidism was investigated by Southern blotting. No detectable alteration in the fragment patterns was observed. Results of segregation analysis in families with affected individuals was inconclusive.

Animals↗
PubMed1990

Cloning and characterization of major antigenic determinants of human cytomegalovirus Ad169 seen by the human immune system.

Starting from a cosmid library of HCMV Ad169-DNA random fragments of DNA were generated. Fragments about 200 to 600 bp in length were selected and cloned into open reading frame (ORF) expression vectors to create ORF-libraries that represent either the entire viral genome or defined subregions. About 120,000 clones were isolated and screened immunologically for the synthesis of fusion proteins consisting of an antigenic peptide encoded by the CMV sequence coupled to a truncated E. coli beta-galactosidase molecule. Anti-CMV sera raised in animals as well as human hyperimmune globulin were used for colony screening. Distinct sets of antigenic fusion proteins were recognized by different antisera. Ten of the clones giving strong reactions with human immune sera were mapped on the CMV genome and the sequences of the CMV inserts determined. Antibodies against fusion proteins were raised in mice or rabbits to identify the corresponding CMV proteins. Antigenic fusion proteins described here were recognized by most individual human CMV immune sera tested. They allow determination of the humoral immune response to defined determinants and may therefore be particularly useful in diagnosis and vaccine development.

Amino Acid Sequence↗
PubMed1990

Large-scale screening of human sera with cytomegalovirus recombinant antigens.

One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulins G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.

Antigens, Viral↗
PubMed1989

Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide.

The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.

Animals↗
PubMed1988

Cosmids.

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Cloning, Molecular↗
PubMed1986

Secretion of glycosylated human interleukin-2 by recombinant mammalian cell lines.

The production of glycosylated forms of the human T cell growth factor (interleukin-2, IL-2) has been studied after transfection of a mouse L cell line and a chinese hamster ovary cell line with a plasmid containing the human chromosomal interleukin-2 gene. Both cell lines produced IL-2 constitutively. Based on their behavior in reversed-phase l.c. and their sodium dodecyl sulfate-gel-electrophoresis pattern, human IL-2 protein secreted by L cells showed a similar distribution of glycosylated (Mr 16 500) and nonglycosylated (Mr 14 500) forms as the natural protein secreted by human peripheral lymphocytes, whereas the hamster cell line secreted preponderantly the glycosylated forms. Exoglycosidase digestion of the 16 500 Mr IL-2 protein shifted the gel electrophoretic mobility towards the low-molecular weight form as is true for the natural glycosylated IL-2, which contains the usual tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-[alpha-NeuAc-(2----6)]-D-GalNAc (IL-2 N2) and the trisaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-D-GalNAc (IL-2 N1) as the major carbohydrate constituents. These results support the applicability of recombinant DNA technology as a tool for studying glycoprotein biosynthesis in mammalian cells.

Animals↗
PubMed1985

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo.

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.

Animals↗
PubMed1979

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.

Animals↗
PubMed1979

Arrangement of coding and intervening sequences of chicken lysozyme gene.

Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene.

Animals↗
PubMed1978

The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322.

In vitro recombination techniques were used to construct hybrid plasmids between pBR 322 and different DNA fragments derived from pML 21 and the E. coli chromosome. Some of the resulting hybrid plasmids express the tetracycline resistance gene of pBR 322, depending on the DNA fragment which has been ligated into the HindIII-site of pBR 322. From these studies we could conclude the direction of transcription of the kanamycin resistance gene in plasmid pML 21 with respect to the SalI, SmaI, HincII, KpnI, EcoRI and HindIII restriction sites. The replacement of the small HindIII-EcoRI-fragment in pBR 322 by other DNA fragments from the E. coli chromosome and selection for tcr-phenotypes showed that this system may be very useful for screening and analysis of promotor-containing DNA fragments.

DNA, Bacterial↗
PubMed1978

Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.

Animals↗
PubMed1977

Transposition and insertion of intact, deleted and enlarged ampicillin transposon Tn3 from mini-R1 (Rsc) plasmids into transfer factors.

The miniR1-(Rsc)-plasmids which derive from the copy mutant R1drd-19B2 (pKN102) are non-conjugative extrachromosomal elements which can not be co-transferred by various transfer factors to recipient strains under standard mating conditions. The attempts to mobilize Rsc11 by F'lac lead to transconjugants carrying F'lac::Tn3 with Tn3 mainly inserted into the lac operon. In addition it can be shown that Rsc11 can become inserted as a complete unit into the transfer factor giving rise to rather unstable recombinant intermediates. Dissociation of these intermediates may lead to alterations of the original plasmids. The Tn3 part of Rsc13 can be enlarged or deleted by in vitro manipulations. In vitro insertion of EcoRI-fragments into an EcoRI+ site of Tn3 leads to new transposable units which can be transposed to the RTF part of R1. This new genetic entity can be stably integrated into the chromosome of E. coli by integrative suppression of a dnaAts-mutation. Deletions at one end or the central region of Tn3 abolish the capability of transposition. However, the Rsc-plasmids containing the deleted Tn3 can still be inserted into the transfer factor as complete units. The resulting recombinants are unstable leading after dissociation in some cases to new plasmids with altered properties.

Ampicillin↗
PubMed1977

Properties of hybrid plasmids, consisting of parts of the mini-R1 factor Rsc11 and ColE1.

In vitro joining of the two small multicopy plasmids Rsc11 and ColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1. Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed. Cleavage of these plasmids with restriction endonucleases HinII, HindIII, EcoRI, SmaI and BamI and hybridization with ColE1 demonstrated that they contain different parts of the parent plasmids, Rsc11 and ColE1. Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid. None of these plasmids can replicate in polA mutants. Replication continues in the presence of chloramphenicol. This suggests that replication can only occur from the ColE1 origin and that the replication function of the Rsc11 part is lost. The hybrid plasmids are compatible with Rsc11 but not with ColE1. The comparison of the physical maps of these Rsc11--ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on the Rsc11 genome.

Ampicillin↗
PubMed1974

Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties.

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.

Centrifugation, Density Gradient↗