Search PubMed⌕ Search

Biomedical subjects

H Lehrach

Publications and source records attributed to H Lehrach.

At least 307 records · Page 17Linked to original sources
PubMed1984

Rapid restriction mapping of DNA cloned in lambda phage vectors.

A protocol for the rapid restriction mapping of phage lambda clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive lambda DNA termini by hybridisation with [32P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the "ladder" of partial digestion products.

Bacteriophage lambda↗
PubMed1984

Selective isolation of cosmid clones by homologous recombination in Escherichia coli.

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.

Animals↗
PubMed1983

Lambda replacement vectors carrying polylinker sequences.

To simplify the construction and screening of genomic libraries, we have made a new family of lambda replacement vectors (EMBL1, EMBL2, EMBL3, EMBL4) and derivatives containing amber mutations (EMBL3 Sam, EMBL3 AamBam, EMBL3 AamSam). These vectors have a large capacity and polylinker sequences flanking the middle fragment. The polylinkers allow a choice of cloning enzymes and, especially useful in the case of cloning of Sau3A partial digests, the excision of the entire insert by flanking SalI (EMBL3) or EcoRI (EMBL4) sites. Phages with inserts can be selected either biochemically (particularly EMBL3) or genetically by their Spi- phenotype. Amber derivatives of the EMBL3 vector allow the application of genetic screening procedures based on selection for the products of homologous recombination events, and for the selective cloning of DNA sequences linked to supF genes.

Animals↗
PubMed1983

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones.

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.

Animals↗
PubMed1982

A procedure to verify an amino acid sequence which has been derived from a nucleotide sequence: application to the 26S RNA of Semliki Forest virus.

We describe a peptide sequencing procedure which can be used to verify an amino acid sequence which is derived from a nucleotide sequence. One first labels the protein with a 3H- and a 14C-labelled amino acid and then cleaves the protein into a set of peptides using a cleavage reaction specific for a particular amino acid residue. Finally one performs Edman degradations on the whole mixture of peptides. The released amino acids reflect the combined aminoterminal amino acid sequences of all the peptides that have been formed by the cleavage reaction. The data can therefore be used to check a deduced sequence simultaneously at several regions of the polypeptide chain. We have applied this sequencing procedure to verify the amino acid sequence deduced from the 26S RNA of Semliki Forest virus.

Amino Acid Sequence↗
PubMed1982

Deletions in immunoglobulin mu chains.

Eight mutant hybridoma lines are described, which synthesize short immunoglobulin mu chains. Four internal deletions were mapped by Southern blot analysis. They are shown to remove DNA from either part or all of the first, and first and second, constant mu exons. The sizes of the deletions range between 0.6 and 5 kb, leaving an equal or unequal number of splice signals. Shorter mu RNA of one size was found irrespective of whether an exon was completely or only partially deleted. These results preclude exclusive 3' (constant region) to 5' (variable region) directional splicing of the mu RNA. No important signals seem to reside in the deleted DNA stretches affecting the transcription or the correct RNA splicing of the remaining exons. The internal mu protein deletions revealed unusual covalent light chain attachment demonstrating functional homology between the first (normally used) and fourth mu constant domain. The other mu protein deletions (10, 11, and 12 kd) involved neither gross DNA nor RNA lesions and are considered to be due to premature chain termination. Since secretion is found in most of the mutant IgM-producing lines, no single one of the four mu constant domains (including the C-terminal one which contains the so-called secretory piece) is necessary for secretion.

Animals↗
PubMed1982

Constant and variable parts in the Balbiani ring 2 repeat unit and the translation termination region.

The large Balbiani rings of the chironomids produce giant internally repeated transcripts that are translated into silk-like proteins used for protective tubes. We have cloned fragments of the Balbiani ring 2 (BR2) gene of Chironomus pallidivittatus, normally the most prominent BR, for sequencing and restriction analysis. The results indicate a basic, tandemly arranged repeat unit of 198 bp, consisting of an invariant region of 102 bp followed by a variable region of 96 bp, the latter containing short internal tandem repeats. In the coding strand of both regions, there is a tendency to maximize adenine ( 40%) and minimize cytosine + thymidine (32-33%). Stringency in codon usage and absence of preference for third position nucleotide substitutions between homologous sequences suggest selection at the nucleotide level to be important. Both regions code for peptides rich in basic amino acids, but show distinct differences in other coding properties. The invariant region probably codes for a crystalline domain, and the variable region for a proline-rich, amorphous domain. One of the clones includes the 3' end of the translated region. Surrounding and following two stop codons is a sequence of four short palindromes. Furthermore, the first stop codon is part of a sequence reminiscent of a "TATA box". The possibilities are discussed that this area of the gene might be a target for regulatory molecules controlling translation termination and/or the expression of an overlapping cistron.

Journal Article↗
PubMed1982

Molecular cloning and expression during myogenesis of sequences coding for M-creatine kinase.

Sequences complementary to muscle poly(A)+RNA were cloned in the plasmid pBR322 and the resulting colonies were screened by colony hybridization with labeled cDNA derived from skeletal muscle and smooth muscle (gizzard). The skeletal muscle-specific clones were further screened by RNA blotting hybridization for a muscle mRNA having the size expected for a putative type M creatine kinase (M-CK) mRNA. The remaining clones with the expected hybridization properties were finally characterized by hybrid-selected translation, and a cloned sequence was shown to contain DNA hybridizing to mRNA that could be translated into M-CK. This plasmid, pMCK1, was further characterized by restriction mapping. Blot analysis of total cell RNA from differentiating myogenic cell cultures showed accumulation of M-CK mRNA in cultures older than 42 hr but not in young little-differentiated cultures.

Animals↗
PubMed1982

Nucleotide sequence at the junction between the nonstructural and the structural genes of the semliki forest virus genome.

The nucleotide sequence at the junction between the nonstructural and the structural genes of the Semliki Forest virus 42S RNA genome has been determined from cloned cDNA. With the aid of S1-mapping, we have located the 5' end of the viral 26S RNA on this sequence. The 26S RNA is homologous to the 3' end of the 42S RNA and is used as a messenger for the structural proteins of the virus. The nucleotide sequence in the noncoding 5' region of the 26S RNA (51 bases) was thus established, completing the primary structure of the 26S RNA molecule (for earlier sequence work, see Garoff et al., Proc. Natl. Acad. Sci. U.S.A. 77:6376-6380, 1980, and Garoff et al., Nature (London) 288:236-241, 1980). An examination of the nucleotide sequences upstream from the initiator codon for the structural proteins on the 42S RNA genome shows that all reading frames are effectively blocked by stop codons, which means that the nonstructural genes in the 5' end of the 42S RNA molecule do not overlap with the structural ones at the 3' end of the molecule.

Base Sequence↗
PubMed1980

A subcloning strategy for DNA sequence analysis.

We describe here a new strategy of fragment preparation for sequencing procedures using endlabelled DNA fragments as substrates (2,3) which is directly applicable to DNA fragments cloned into the Pst I site of pBR322, or in modified form, to inserts into the BamH I or Sal I site of the same plasmid. Ordered sets of subclones of predetermined overlap are are generated. These can be sequenced directly without further strand- or fragment separation steps.

Base Sequence↗
PubMed1980

Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids.

Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

Actins↗
PubMed1980

The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region.

The amino acid sequence of the capsid (C) protein was deduced from the nucleotide sequence of the C gene. This part of the viral 42S RNA genome was transcribed into double-stranded cDNA. The cDNA was cloned in the Escherichia coli chi 1776-pBR322 host-vector system and then the base sequence was determined with the technique described by Maxam and Gilbert. The amino acid sequence of the C protein shows a clustering of basic amino acids and prolines within the first 110 amino acids.

Amino Acid Sequence↗
PubMed1980

Construction of recombinant plasmids containing rat muscle actin and myosin light chain DNA sequences.

The construction and partial characterization of recombinant bacterial plasmids carrying DNA sequences that hybridize with rat skeletal muscle actin and a myosin light chain mRNA is described. DNA of one clone hybridizes specifically with the muscle-specific alpha-actin mRNA. Three plasmid clones contain DNA inserts that hybridize with muscle as well as with nonmuscle actin mRNA. A fifth plasmid contains sequences complementary to mRNA coding for myosin light chain 2. DNA of this plasmid hybridizes specifically with RNA extracted from muscle and differentiated muscle cultures but not with RNA extracted from proliferating mononucleated myogenic cells.

Actins↗
PubMed1979

Directive segregation in the basis of colE1 plasmid incompatibility.

Incompatibility between colE1 plasmids in Escherichia coli can be explained by competition for a limited number of replication sites. These sites ensure directive segregation of plasmids to daughter cells on cell division. The number of sites can be more than one only if a linear segregation mechanism is postulated.

Bacteriocin Plasmids↗