Lambda phage vectors--EMBL series.
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Bacteriophage lambda↗
Biomedical subjects
H Lehrach
Publications and source records attributed to H Lehrach.
DNA sequencing using alpha-thiodeoxynucleotides.
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Base Sequence↗
A family of cosmid vectors with the multi-copy R6K replication origin.
A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.
Bacteriophage lambda↗
Tyrosine aminotransferase and chymotrypsinogen B are linked to haptoglobin on human chromosome 16q: comparison of genetic and physical distances.
The loci for haptoglobin (HP) and tyrosine aminotransferase (TAT) are known to reside at 16q22. Chymotrypsinogen B (CTRB), which is syntenic with TAT and HP on mouse chromosome 8, has also been assigned to human chromosome 16 but has not been mapped regionally. A linkage analysis was carried out in 13 informative families using RFLPs for these three markers. For CTRB, two TaqI RFLPs with a polymorphism information content of 0.60 derived from haplotype frequencies are described. The most likely order of loci, deduced from triple informative crosses, and their map distances, obtained by pair-wise linkage analysis, are HP-7 cM-TAT-9 cM-CTRB. By pulsed-field gel electrophoresis, a physical map covering more than 2000 kb was constructed. A maximum physical distance of about 700 kb was obtained for HP and TAT, which contrasts with the genetic distance of 7 cM (approximate confidence limits 2-18 cM). CTRB is at least 800 kb away from these two markers.
Amino Acid Metabolism, Inborn Errors↗
Molecular approaches to genome analysis: a strategy for the construction of ordered overlapping clone libraries.
Here we describe progress on a series of molecular techniques designed to bridge the gap between genetic and molecular distances in mammals. This is an essential step in the molecular cloning of genes defined by mammalian mutations, and in the molecular analysis of large regions of mammalian genomes. We summarize approaches for the physical and molecular analysis of genetic distances and describe the experimental, statistical and computational basis of a new approach to create ordered libraries of overlapping clones from large genomes.
Bayes Theorem↗
Molecular evidence for the rapid propagation of mouse t haplotypes from a single, recent, ancestral chromosome.
Mouse t haplotypes are variant forms of chromosome 17 that exist at high frequencies in worldwide populations of two species of commensal mice. To determine both the relationship of t haplotypes to each other and the species within which they exist, 35 representative t haplotypes were analyzed by means of 10 independent molecular probes, including five DNA clones and five polypeptide spots identified by means of two-dimensional gel electrophoresis. All of the tested haplotypes were found to share restriction fragments and polypeptide spots that are absent in mice carrying wild-type forms of chromosome 17. This observation provides the first direct evidence that all of the known t haplotypes are descendents of a single ancestral chromosome. The absence of variation among t haplotypes could mean that this ancestral chromosome existed relatively recently, in which case it would be necessary to postulate introgressions of t haplotypes across species lines to explain their presence in both Mus domesticus and M. musculus. Alternatively, it is possible that the ancestral chromosome existed prior to the split between M. domesticus and M. musculus and that, by chance, our probes fail to detect polymorphisms that exist among the t haplotypes. A further result of our analysis is the characterization of a partial t haplotype in a wild population of Israeli mice.
Animals↗
Deletion and duplication of DNA sequences is associated with the embryonic lethal phenotype of the t9 complementation group of the mouse t complex.
We have analyzed the genomic structure of three mouse t haplotypes of the t9 complementation group. Each of these t haplotypes, tw18, t4, and tks1, is known to have resulted from a rare recombination event between a complete t haplotype and a wild-type chromosome. Using molecular probes that identify sequences in the distal portion of the t complex, we have shown that each of these t haplotypes contains a similar (perhaps identical) deletion of one group of t complex sequences, and duplication of another group. These data suggest that the recombination events that produced these three t haplotypes involved similar unequal crossovers within the distal inversion. The deletion and duplication of genetic material associated with all members of the t9 complementation group tested provides a molecular explanation for the recessive lethal mutation associated with these t haplotypes.
Animals↗
Genetic analysis of the proximal portion of the mouse t complex: evidence for a second inversion within t haplotypes.
Genomic sequences derived from the mouse t complex by a microdissection cloning technique have been used as tools to obtain high resolution genetic maps of the wild-type and t haplotype forms of the most proximal portion of chromosome 17. Genetic mapping was performed through a recombinant inbred strain analysis and an analysis of partial t haplotypes. The accumulated data demonstrate the existence of a large inversion of genetic material, encompassing the loci of T and qk, within the proximal portion of t haplotypes. This newly described proximal inversion and the previously described distal inversion provide an explanation for the suppression of recombination observed along the length of t haplotype DNA in heterozygous mice.
Animals↗
A computer program package for restriction map analysis and manipulation.
Programs for the calculation, storage and analysis of restriction maps derived from the analysis of partial digestion products from end labelled DNA (1,2,3) and their correlation with digestion - and hybridisation patterns in total digestions and Southern blot experiments are described. These programs allow direct input of gel patterns from partial or complete digestion experiments using a digitizer tablet, calculation of molecular weights and restriction maps, plotting of maps and actual or predicted fragment patterns and automated identification of overlapping cosmids from partial restriction mapping results. Programs are written in PASCAL and have been implemented on a VAX/VMS system, with a HP-7221T plotter and a digitizing tablet.
Cloning, Molecular↗
Genetic and cytogenetic localisation of the homeo box containing genes on mouse chromosome 6 and human chromosome 7.
Probes from the m6 homeo box cluster were mapped to mouse chromosome 6 by somatic cell genetics, in situ hybridisation, and by a Mus spretus--Mus musculus backcross mapping system. In addition, the testis-specific homeo box containing cDNA, clone, HBT-1, has been mapped using the same back-cross system and the B X D recombinant inbred strain set. Close genetic and physical linkage between the m6 cluster and HBT-1 was demonstrated, positioning these sequences to the same local cluster of homeo box containing genes. The map location of this cluster between IgK and Tcrb coincides with the morphological mutation hypodactyly (Hd). Synteny has been observed between a region of mouse chromosome 6 and the long arm of human chromosome 7 encompassing the markers Cpa, Tcrb and Try-1. Here we localise human sequences hybridising to the mouse m6 probes to the short arm of chromosome 7, breaking the region of synteny.
Animals↗
A new method of DNA sequencing using deoxynucleoside alpha-thiotriphosphates.
A new procedure for determining DNA nucleotide sequences is reported. In the first step of the method, four DNAs, each separately substituted with a different deoxynucleoside phosphorothioate in place of the corresponding monophosphate, are prepared by template-directed polymerization catalyzed by DNA polymerase. In the second step, these DNAs are subjected to stringent exonuclease III treatment, which produces only fragments terminating with a phosphorothioate internucleotide linkage. These can then be separated by standard gel electrophoresis techniques and the sequence can be read directly as in presently used sequencing methods.
Base Sequence↗
Molecular approaches to mammalian genetics.
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Animals↗
Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene.
The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.
Animals↗
Molecular probes define different regions of the mouse t complex.
Four genomic clones obtained from microdissected fragments of the proximal portion of mouse chromosome 17 have been used to identify a series of t-haplotype-specific restriction fragments. Their specificity is defined by presence in eight complete t haplotypes and absence from 18 inbred strains of wild-type mice. Partial t haplotypes contain subsets of the t-specific fragments, and each can be classified according to the t-specific fragments it contains. This is the first molecular evidence that independent partial t haplotypes contain different lengths of t haplotype DNA. Recombination studies indicate that partial t haplotypes suppress recombination in proportion to the extent of t haplotype DNA they contain. Molecular analysis of partial t haplotypes shows that the t-specific fragments map to and thus define different regions of the t complex. Certain regions of t haplotype DNA defined by t-specific restriction fragments can be correlated with loci involved in the control of transmission ratio distortion.
Alleles↗
Analysis of cosmids using linearization by phage lambda terminase.
A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.
Animals↗
The structure of a cloned mouse gamma-actin processed pseudogene.
The complete nucleotide (nt) sequence of a gamma-actin-like pseudogene (M gamma A-psi 1), isolated from a mouse genomic library in phage lambda, was determined. The pseudogene was shown to be of the processed type by the fact that it lacked introns, ended in a poly(dA) region, and was flanked by direct repeats. There were ten differences in the predicted amino acid (aa) sequence from that of the authentic nonmuscle gamma-actin. An unusual feature of M gamma A-psi 1 was the complete absence of DNA corresponding to the 5' end of the mRNA up to the nt preceding the Ala codon at aa position 7. This suggests that M gamma A-psi 1 originated from a truncated mRNA or from an incomplete reverse transcript.
Actins↗
Molecular tools for the mapping of the human genome.
The rapid progress in molecular cloning and DNA analysis techniques, together with the use of cloned DNA probes from specific chromosomes of the human genome might allow localisation and ultimately identification of genes defined by single mutations. The discrepancy between genetic dimensions expressed in centimorgans, each corresponding to millions of base pairs and distances easily accessible by molecular techniques amounting to at the most hundreds of kilobase pairs may be bridged with some new cloning techniques partially developed at the European Molecular Biology Laboratory. These techniques were designed to allow rapid cloning and analysis of large regions of mammalian genomes.
Animals↗
Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes.
Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.
Animals↗