Recombination-Based screening for genes on chromosome 21

In previous work, a reasonably large number of genes was found to be on chromosome 21. This frequency demands the creation of new techniques to investigate the transcription of this small human autosome, and to relate these findings to the phenotype of Down syndrome. Here we describe the elaboration of new vectors and hosts, which in conjunction with flow-sorted cosmid libraries of chromosome 21 and genic (cDNA) libraries of relevant human tissues, will permit us to examine transcription of chromosome 21.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38253/1/1320370723_ftp.pd

A small plasmid for recombination-based screening

We reported recently the construction of the 4.4-kb RoK-derived pMADI plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with Co1E1 and therefore permits recombination-based screening of 2 libraries that contain Co1E1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the [pi]-encoding pir gene required for R6K replication. To supply [pi] [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans we employed pPR1[Delta]22pir 116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger 2 inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1(1991) 120-123].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29693/1/0000025.pd

The P2 phage old gene: sequence, transcription and translational control

The old (overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage [lambda] growth [ Sironi et al., Virology 46 (1971) 387-396 ; Lindahl et al., Proc. Natl. Acad. Sci. USA 66 (1970) 587-594]. Specialized transducing [lambda] phages, which lack the recombination region, can be selected by plating [lambda] stocks on E. coli that carry the old gene on a prophage or plasmid [Finkel et al., Gene 46 (1986) 65-69]. Deletion and sequence analyses indicate that the old-encoded protein has an Mr of 65 373 and that its transcription is leftward. Primer extension analyses locate the transcription start point near the right end of the virion DNA. A bacterial mutant, named pin3 and able to suppress the effects of the old gene, has been isolated [Ghisotti et al., J. Virol. 48 (1983) 616-626]. In a pin3 mutant strain, carrying the old gene on a prophage or plasmid, the amount of old transcript is greatly reduced. The effect of the pin3 mutation is abolished by the wild-type allele of argU, an arginine tRNA that reads the rare Arg codons AGA and AGG, which are used for eight of the 14 Arg codons in the old gene. Thus the pin3 allele probably stalls translation of the old mRNA, causing this mRNA to be degraded. Isoelectric focusing and electrophoretic analysis identify the old gene product as a basic protein of approx. 65 kDa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27631/1/0000007.pd

Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21

Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid [beta] protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29821/1/0000167.pd

Plasmids for recombination-based screening

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, [pi]G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29122/1/0000161.pd

Isolation of DNA sequences on human chromosome 21 by application of a recombination-based assay to DNA from flow-sorted chromosomes

By merging two efficient technologies, bivariate flow sorting of human metaphase chromosomes and a recombination-based assay for sequence complexity, we isolated 28 cloned DNA segments homologous to loci on human chromosome 21. Subregional mapping of these DNA segments with a somatic cell hybrid panel showed that 26 of the 28 cloned DNA sequences are distributed along the long arm of chromosome 21, while the other 2 hybridize with sequences on the short arm of both chromosome 21 and other chromosomes. This new collection of probes homologous to chromosome 21 should facilitate molecular analyses of trisomy 21 by providing DNA probes for the linkage map of chromosome 21, for studies of nondisjunction, for chromosome walking in clinically relevant subregions of chromosome 21, and for the isolation of genes on chromosome 21 following the screening of cDNA libraries.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47623/1/439_2004_Article_BF00366237.pd

The murine situs inversus viscerum (iv) gene responsible for visceral asymmetry is linked tightly to the Igh-C cluster on chromosome 12

The iv gene controls left-right determination during murine organogenesis. To map this gene, we analyzed backcross progeny produced by mating (C57BL/6J x MEV/Ty)F1-iv/+ heterozygotes to C57BL/6J-iv homozygotes. Hybridization of a murine ecotropic virus probe and several homeotic box gene probes coupled with analysis of dominant visible markers enabled us to exclude the iv locus from much of the mouse genome. Spurred by a recent report that mapped the iv gene to mouse chromosome 12 which was not excluded by our previous work, we used the polymerase chain reaction on our larger cohort to determine that the iv gene is indeed linked tightly to the Igh-C locus on this chromosome: we observed 0/156 recombinants between the iv and Igh-C loci. Combining data from the two studies demonstrates that the murine iv gene is close (1/201 recombinants) to the Igh-C cluster on chromosome 12.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28508/1/0000305.pd

Escherichia coli recA deletion strains that are highly competent for transformation and for in vivo phage packaging

I describe the construction of a variety of Escherichia coli recA deletion strains designed to facilitate molecular cloning. These recA deletion strains permit the efficient cloning of foreign inserts carried in plasmid, phage, cosmid, phasmid (phage-plasmid hybrid) or phosmid (phage-cosmid hybrid) vectors. (Bacteriophage A; cosmids; phasmids; phosmids; plasmid vectors; recombinant DNA)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27713/1/0000101.pd

A rapidly-banding component of animal DNA preparations that interferes with buoyant density studies

Rapidly-banding components of purified animal cell DNA's have been observed which have abnormal buoyant densities and show anomalous band profiles when centrifuged to equilibrium in cesium salts. These components interfere with the separation of animal DNA fractions and with the analysis of animal cell DNA's by buoyant density techniques. They can be detected by examining photographs of analytical buoyant-density ultracentrifugations before equilibrium has been reached. These rapidly-banding components are removed by phenol treatment