Rosann A. Farber

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication.

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the neardiploid mouse CAK cell line have been analyzed. The Aprt gene is located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from whichAprt− mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele, with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detcted in cell lines lacking morphologically marked chromosomes.

Ninety-Six Haploid Yeast Strains With Individual Disruptions of Open Reading Frames Between YOR097C and YOR192C, Constructed for the Saccharomyces Genome Deletion Project, Have an Additional Mutation in the Mismatch Repair Gene MSH3

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene.

Cytological localization of adenosine kinase, nucleoside phosphorylase-1, and esterase-10 genes on mouse chromosome 14

We have determined the regional locations on mouse chromosome 14 of the genes for mouse adenosine kinase (ADK), nucleoside phosphorylase-1 (NP-1), and esterase-10 (ES-10) by analysis of rearranged mouse chromosomes in γ-irradiated Chinese hamster × mouse hybrid cell lines. Irradiated clones were screened for expression of the murine forms of these enzymes; segregant clones that expressed only one or two of the three markers were karyotyped. The patterns of enzyme expression in these segregants were correlated with the presence of rearranged chromosomes. The Adkgene was localized to bands A2 to B, Np-1to bands B to C1, and Es-10to bands D2 to E2.

Relationships among DNA sequences of the 1.3 kb EcoRI family of mouse DNA.

SummaryThe genome of the mouse (Mus musculus) contains a family of repeated DNA sequences defined by a 1.3 kb EcoRI fragment. Resqtriction maps of ten cloned fragments from this family have been determined. The fragments were of seven different types, based on the patterns of digestion obtained with AvaII, HindIII, and TaqI restriction enzymes. These seven unique sets of sequences fell into two classes, as defined by the position of a single HindIII site. Portions of fragments from each of the two classes were sequenced. Although certain regions of the repeat were highly conserved between classes, there was more intraspecific sequence divergence among the sequenced regions than has been observed for the short interspersedAlu family of repeated sequences sin mammals. Sequences of both HindIII classes were found to be present within the mouse X chromosome; we can conclude that both classes must also be present on other mouse chromosomes.

Patterns of replication of human chromosomes in human x mouse hybrids with different chromosomal compositions

The order of termination of DNA replication of ten human chromosomes remaining in a hybrid between normal human skin fibroblasts and mouse RAG calls has been analyzed. The human chromosomes were Nos. 2, 3, 4, 5, 6, 7, 10, 16, 17 and the Y. The order of replication of these chromosomes was essentially the same as that of the corresponding chromosomes in normal fibroblasts. This hybrid contained four human chromosomes, 6, 16, 17, and the Y, which were not present in a related hybrid (RRP5-4) which had been studied previously. Several chromosomes in RRP5-4, including 4, 5, and 7, had been shown to replicate at different times than the same chromosomes in the normal parental fibroblasts. These results suggest that there may be specific genes which are important for the control of the precise order of replication of human fibroblast chromosomes. These genes could be located on chromosomes which were retained in the hybrid analyzed here but which were missing from RRP5-4.

Somatic cell hybrid mapping of human chromosome band 5q31: A region important to hematopoiesis

As a means of characterizing the distal long arm of chromosome 5, in particular, the region spanning 5q23-->q31, we analyzed somatic cell hybrids prepared from cells with overlapping chromosomal rearrangements. In one hybrid, the derivative chromosome 5 from a patient with acute myeloid leukemia (AML) de novo, whose bone marrow cells had a balanced translocation, t(5;7)(q31;q22), involving chromosome band 5q31, was isolated in a somatic cell hybrid (B294). In addition, we prepared somatic cell hybrids from a lymphoblastoid cell line (CC) derived from a patient who has a constitutional interstitial deletion of chromosome 5 spanning 5q23.1-->q31.1. By a combination of Southern hybridization analysis and fluorescent in situ hybridization, we constructed a map dividing 5q23-->q31 into four regions. We can assign genes to these regions and relate them to anonymous RFLP markers that have been genetically mapped.

Chromosome replication in aging human diploid fibroblasts

Abstract The patterns of termination of DNA replication in human embryonic MRC-5 fibroblasts at four passage levels have been examined by autoradiography. Only chromosome 9 showed statistically significant differences in the time of replication among cultures of different ages. This chromosome terminated replication earlier at later passages than at earlier passages, primarily because of differences in the time of replication of the centromere region. Because very few differences were observed at different passage levels, we conclude that changes in the order of chromosome replication are unlikely to contribute to the phenomenon of in vitro senescence.

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.

Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.

Chromosome replication in normal and transformed human lymphocytes

We analyzed the late-replication patterns of human B-lymphocyte chromosomes before and after transformation by Epstein-Barr virus. There were no statistically significant differences between normal cells and transformed cells derived from the same male individual; therefore, the order of termination of chromosome replication was unchanged by transformation. We also examined the replication patterns of T lymphocytes from the same donor and found no differences between normal B and T cells.