Biomedical subjects
R H Reeves
Publications and source records attributed to R H Reeves.
Yeast artificial chromosome modification and manipulation.
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Colony-hybridization apparatus with batch-processing capacity.
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High-efficiency yeast artificial chromosome fragmentation vectors.
Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.
Integrative selection of human chromosome-specific yeast artificial chromosomes.
Human specific "integrative selection vectors" (ISVs) were designed to optimize integration of a yeast-selectable marker specifically into yeast artificial chromosomes (YACs) derived from human but not mouse DNA. ISVs were transformed into a YAC genomic library constructed from DNA of a human-mouse somatic cell hybrid containing chromosome 21 (HSA21) as the only human chromosome. One percent of the yeast in the original library contained HSA21-derived YACs; between 45% and 54% of the yeast recovered after transformation with ISV vectors contained human YACs. Integrative selection provides a rapid means of obtaining a highly enriched population of human chromosome-specific YACs by eliminating the labor-intensive steps of isolating and screening primary transformants. The procedure is biased toward the selection of YACs that contain a large number of targets for homologous recombinations; thus, libraries constructed by this procedure will be composed primarily of the largest YACs in the population.
Comparison of interspecific to intersubspecific backcrosses demonstrates species and sex differences in recombination frequency on mouse chromosome 16.
One hundred fourteen progeny from an interspecific backcross between laboratory mice and M. spretus were typed for six markers spanning most of mouse Chromosome (Chr) 16. Additional maps of 9-10 markers of this chromosome were derived from analysis of over 500 progeny from four backcrosses between inbred laboratory strains and members of the Mus musculus group, M.m. musculus and M.m. molossinus (subspecies). The results of these analyses confirmed the gene order: (CEN)-Prm-1/Prm-2-Igl-1-Smst-Mtv-6-Gap43-Pit-1(dw)- D21S16h-App-Sod-1-Ets-2-Mx. Maps produced from these five crosses were of similar lengths, but recombination in several regions was affected by sex of the F1 parent or by the combination of strains used in the cross. As reported previously, recombination frequencies were elevated significantly at the distal end of the chromosome in a cross using F1 males. The male map showed significant compression in the interval Smst to Gap43. Both male and female intersubspecific maps were expanded near the proximal and distal ends of the chromosome relative to the interspecific cross. The spretus cross was compressed in the proximal interval, Prm-1-Igl-1-Smst, and was slightly expanded in the Smst-Gap43 interval, relative to intersubspecific crosses using F1 females. Female intersubspecific maps were expanded about 50% near the distal end of the chromosome when compared to the interspecific cross. The expansion or compression of maps using different strain or sex combinations has implications for the efficient production of high resolution recombinational maps of the mouse genome.
Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster.
Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters.
Modification and manipulation of mammalian DNA cloned as YACs.
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The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation.
Two nonallelic mouse mutations with severe dwarf phenotypes are characterized by a lack of growth hormone, prolactin, and thyroid stimulating hormone. The cells that normally synthesize these pituitary hormones express a common transcription factor called GHF-1 or Pit-1. Using an intersubspecific backcross, we have demonstrated tight linkage of the Pit-1 and Snell dwarf (dw) genes on mouse chromosome 16. No recombination was observed between Pit-1 and dw in 110 individuals examined. Southern blot analysis of genomic DNA reveals that the Pit-1 gene is rearranged in C3H/HeJ-dwJ/dw mice but not in coisogenic +/+ animals, providing molecular evidence that a lesion in the Pit-1 gene results in the Snell dwarf phenotype. Demonstration of low levels of Pit-1 expression in Ames dwarf (df) mice implies that both Pit-1 and df expression may be required for pituitary differentiation.
Sex, strain, and species differences affect recombination across an evolutionarily conserved segment of mouse chromosome 16.
A region of substantial genetic homology exists between human chromosome 21 (HSA21) and mouse chromosome 16 (MMU16). Analysis of 520 backcross animals has been used to establish gene order in the homologous segment. D21S16h and Mx are shown to represent the known proximal and distal limits of homology between the chromosomes, while Gap43, whose human homolog is on HSA3, is the next proximal marker on MMU16 that has been mapped in the human genome. Recombination frequencies (RFs) in four intervals defined by five loci in the HSA21-homologous region of MMU16 were analyzed in up to 895 progeny of eight different backcrosses. Two of the eight crosses were made with F1 males and six with F1 females. The average RF of 0.249 in 265 backcross progeny of F1 males was significantly higher than the 0.106 average recombination in 320 progeny of F1 females in the interval from D21S16h to Ets-2. This is in contrast to HSA21, which shows higher RFs in female meiosis in the corresponding region. Considerable variation in RF was observed between crosses involving different strains, both in absolute and in relative sizes of the intervals measured. The highest RFs occurred in a cross between the laboratory strain C57BL/6 and MOLD/Rk, an inbred strain derived from Mus musculus molossinus. RFs on this cross were nearly fivefold higher than those reported previously for an interspecific cross between C57BL/6 and Mus spretus.
Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes.
Mammalian DNA segments cloned as yeast artificial chromosomes (YACs) can be manipulated by DNA-mediated transformation when placed in an appropriate yeast genetic background. A "fragmenting vector" has been developed that can introduce a yeast telomere and selectable marker into human-derived YACs at specific sites by means of homologous recombination, deleting all sequences distal to the recombination site. A powerful application of the method uses a human Alu family repeat sequence to target recombination to multiple independent sites on a human-derived YAC. Sets of deletion derivatives generated by this procedure greatly facilitate restriction mapping of large genomic segments. Targeting recombination with single copy sequences, such as cDNAs, will have many additional applications. This approach establishes a paradigm for manipulation and characterization of mammalian DNA segments cloned as YACs.
Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome.
A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.
Developmental expression of the amyloid precursor protein, growth-associated protein 43, and somatostatin in normal and trisomy 16 mice.
The expression during development of 3 genes located on mouse chromosome 16 (MMU 16) which are implicated in neurobiological processes was examined by blot hybridization beginning at early gestational ages in the mouse. The 3 genes, amyloid precursor protein (App), preprosomatostatin (Smst), and growth-associated protein 43 (Gap43), exhibited distinct profiles of expression. App expression increased steadily throughout fetal and postnatal development. Smst expression peaked during the third postnatal week, then reached a plateau at a slightly lower level in adults, and Gap43 expression was highest in the early postnatal period, declining in adults to levels below those seen at the earliest timepoints examined. Smst message levels exhibited a 1.5-fold increase in the brains of trisomy 16 (Ts16) mice as compared to normal littermates on day 15 of gestation, while Gap43 and App message levels were elevated approximately 2-fold.
The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21.
Utilizing the backcross C57BL/6 wv/wv x (C57BL/6 wv/wv x MOLD/Rk), the mouse neurological mutation weaver (wv) was mapped less than 1 cM proximal to Ets-2 and Mx on mouse chromosome 16 (0.96 +/- 0.1% recombination). This region is known to include eight genes that are found on human chromosome 21 (HSA 21) and appears to be highly conserved between the two species. We therefore predict that the normal human homolog of wv will be located on HSA 21 and would be in dosage imbalance in individuals with Down syndrome.
Down syndrome, Alzheimer's disease and the trisomy 16 mouse.
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Genetic mapping and analysis of somatostatin expression in Snell dwarf mice.
Mice homozygous for the gene dwarf (dw) have elevated levels of somatostatin (SS) in extra-hypothalamic brain regions. By in situ hybridization, increased levels of SS mRNA were observed in regions shown previously to contain higher levels of the SS peptide. Thus, the rate of transcription and/or the stability of SS mRNA are affected by the dw mutation. Since both dw and the gene encoding SS, Smst, are located on mouse chromosome 16, two backcrosses segregating dw and Smst were used to determine whether dw is an allele of Smst. In one backcross, an inbred strain derived from the subspecies Mus musculus molossinus was used to provide a high degree of DNA sequence polymorphism. The gene order and map distances determined on this backcross were: (centromere) - Prm-1 - 7 - Igl-1 - 3 - Smst - 29 - dw - 15 - Sod-1 - 4 - Ets-2, demonstrating clearly that Smst and dw are distinct genes. Additional evidence against a primary role for SS excess in the pathogenesis of dw/dw mice was obtained by injecting normal newborn mice with a potent SS analog (cyclo II). In contrast to the pattern of cell loss observed in the dwarf anterior pituitary, the pituitaries of injected mice were indistinguishable from normal controls, further suggesting that the Smst locus is not the primary site of dw gene action.