Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.     

Microclones from a mouse germ line HSR detect amplification and complex rearrangements of DNA sequences.

The DNA sequence organization of a homogeneously staining region (HSR) in the germ line of Mus musculus was studied with DNA clones generated by microdissection and microcloning. Six HSR-derived microclones were selected and characterized by Southern blot hybridizations. Four represented single-copy mouse DNA sequences. They were amplified in the HSR as fragments co-migrating with the respective normal mouse sequence and as additional fragments of different mobilities. The copy number of co-migrating fragments was approximately 16 for each of the four sequences but the number of rearranged fragments varied. Two microclones contained DNA sequences not detectable in normal mouse genomes but present, and one of them amplified, in the HSR. The observations suggest that the HSR developed from a part of the mouse genome by alternating replication and rearrangement events, with a specific integration of putative foreign DNA sequences.     

Strange goings-on in the mouse germ line

It is a conventional paradigm that mutagens lead to changes in nucleotide sequence when the cell attempts to repair or replicate lesions in DNA (such as adducts or strand breaks) that have been produced by the mutagens or their metabolites. The resulting changes are located at (or very near) the sites of the initial damage. This is the underlying theory behind mutational spectra work, but how general is it in vivo? Work with ionising radiation has shown that there are interesting things going on in the mouse germ line that do not fall within the conventional paradigm. Mutations occur at certain sites remote from initial DNA damage and in greater than expected number. Bryn Bridges discusses some recent papers on mutational changes in the germ line of mice following exposure to chemical mutagens that suggest that such phenomena may not be confined to radiation.     

Localization of the multidrug resistance-associated 170 kDa P-glycoprotein gene to mouse chromosome 5 and to homogeneously staining regions in multidrug-resistant mouse cells by in situ hybridization

This report describes the localization of the 170 kDa P-glycoprotein gene(s) to mouse chromosome 5, subbands A2 or A3. Overproduction of P-glycoprotein is associated with multidrug resistance (MDR). MDR cell lines derived from the SEWA mouse tumor carry multiple copies of the P-glycoprotein gene. These were found to reside in homogeneously staining regions, situated in different locations in different sublines.     

Evidence for noncontiguous variable and constant region genes in both germ line and myeloma DNA.

A study has been made of the hybridization of mouse light chain cDNA to restriction enzyme digests of DNA. DNA was digested with B. st (specificity GGATCC) and fractionated on preparative agarose gels for hybridization analysis. Experiments with liver or kidney DNA yielded two peaks of hybridization with V+C cDNA corresponding to the C_κ gene and to the germ line V gene homologous to the MOPC21 V gene. Since there is no site for digestion by B. st within the MOPC21 V or C genes, this result shows that the germ line DNA carries separately the V²¹ and C_κ genes.The hybridization profiles of two different myeloma DNAs (MOPC21 and AdjPC5) differed from those of the germ line DNA. In both myelomas, only one hybridization peak was observed and no peaks corresponding to the germ line pattern were seen. The new pattern of hybridization implies that the events involved in maturation of antibody-producing cells includes rearrangement of the V and C genes.To study whether this proposed rearrangement of DNA results in contiguous V and C genes in producing cells, discrete V+C cDNA size classes (prepared with MOPC21 mRNA) were hybridized to both unfractionated restriction digested MOPC21 DNA and to the partially purified L chain gene of MOPC21 DNA. The length of the cDNA rendered resistant to single-strand-specific S₁ nuclease was determined. In no case was the full length of V+C cDNA protected from nuclease; instead, a fragment of about 290 bases (C region length) plus smaller fragments were generated. These results indicate that the rearrangement of L chain genes, which seems to occur in myeloma cells, may well not produce contiguous V and C genes in the DNA.     

A long-range repeat cluster in chromosome 1 of the house mouse, Mus musculus, and its relation to a germline homogeneously staining region.

The laboratory mouse C57BL genome contains about 50 copies of a long-range repeat DNA family clustered in the C-D region of chromosome 1. The repeat length is more than 50 kb and includes sequences homologous to at least two mRNAs. There are small differences in the copies of this repeat family such as restriction site mutations and gross differences like rearrangements and insertions of LINE1 elements. A germline homogeneously staining region occurring as a chromosome 1 polymorphism in many feral populations of the house mouse is an amplified version of this long-range repeat cluster.     

A structurally defined mini-chromosome vector for the mouse germ line

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines

The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.     

Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre

Cell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoter-driven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at approximately e15, and was >95% efficient in male and female germ cells by birth. Although there was a potent maternal effect with some animals showing more widespread recombination, there was no ectopic activity in most adults. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of gametogenesis and as a general deletor line.     

Loss of irreversibility of granulocytic differentiation induced by dimethyl sulfoxide in HL-60 sublines with a homogeneously staining region.

The human HL-60 acute leukemia cell line harbors double minutes (dmins) during early passages. During its continuous culture for a long term, a single marker chromosome with a homogeneously staining region (HSR) replaces the dmins. The both structures harbor amplified c-MYC sequences. Here we ask how the cellular phenotype is altered by the c-MYC integration into a HSR. Treatment with dimethyl sulfoxide induces granulocytic differentiation in the both types of cells. In contrast to HL-60/dmin cells, however, no apoptosis followed differentiation and the differentiation phenotype was reverted upon withdrawal of the drug in HL-60/HSR cells. Terminal differentiation and loss of DNase I hypersensitivity sites at c-MYC P2 promoter appeared to be unlinked in the both types of cells. By comparison with HL-60/dmin cells, we conclude that the integration into a HSR of an extrachromosomal gene(s) but not c-MYC likely leads to the loss of irreversibility of the differentiation phenotype.     

Correlation of unstable multidrug cross resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1.

An enrichment selection method using repeated pulses of low drug concentration (1 microgram/ml) was used to isolate CHO (AK412) variants that are 20-fold more resistant to cytochalasin D (CD). CD-resistant (CydR) variants possess a unique unstable phenotype, including a longer doubling time in nonselective medium, a higher frequency of multinucleate cells in the population (probably due to a defect in cytokinesis), an altered morphology, and increased resistance or sensitivity to a number of unrelated drugs. In each of two variant lines examined cytologically, this multiple phenotype is associated with a small homogeneously staining region on chromosome 1. The homogeneously staining region is present in the CydR variants, but absent both in the CD-sensitive parent and in a CD-sensitive revertant subpopulation. Studies of CD-displaceable binding of [3H]cytochalasin B show a fourfold reduction in CD binding or uptake when whole cells of the variant line were examined. Lactoperoxidase-catalyzed iodination and metabolic labeling with [H3]fucose of cell surface proteins of the CydR variants showed multiple differences in electrophoretic band migration when compared with parental proteins.     

Transposition of the Drosophila hydei Minos transposon in the mouse germ line

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.     

Maintenance of mouse male germ line stem cells in vitro

The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. We analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. Although the number of adult and immature SSCs decreased in a time-dependent manner, nearly one in four stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or utilization of specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines and addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained when cultured with TM4 cells or activin A, and 6% remained when cultured with SF7 cells or BMP4. These results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.     

Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line

The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions.     

Evidence for a fifth (G) region in theH-2 complex of the mouse

Antisera (B10.129×A)F₁ anti-P and (B10×A)F₁ anti-B10.P contain antibodies that define, in the PVP hemagglutination test, an antigen originally described as G or H-2.7. Of the independentH-2 haplotypes, the H-2.7 antigen is present inf, j, k, p, ands. In addition, the antisera also contain a weak cytotoxic antibody, distinct from anti-H-2.7. The cytotoxic antibody reacts with antigens controlled by theK orI regions. The hemagglutinating H-2.7 antibody does not have cytotoxic activity. The genetic determinant coding for antigen H-2.7 can be mapped into the chromosomal segment between theS andD regions. The H-2.7 antigen thus serves as a marker for a new region of theH-2 complex. The locus coding for antigen H-2.7 is designatedH-2 G and the correspondingH-2 regionG. The H-2.7 antigen has a tissue distribution distinct from that of the H-2 antigens controlled by theK orD regions. So far it could be detected primarily on erythrocytes.