Human Repetitive DNA Sequences Research Articles

An efficient chemical method to generate repetitive sequences depleted DNA probes

We describe an efficient method to remove repetitive sequences from DNA of microdissected whole chromosomes, chromosome arms, locus specific probes, and specific bands. The chemical approach described removes human repetitive DNA sequences from a source DNA, eliminating the need for blocking such sequences when the source DNA used as a probe is hybridized with a target DNA. It employs subtracting hybridized biotin-labeled repetitive-sequence DNA complex with phenol and chloroform after incubation of hybridized products with avidin. The method produces unique products that are formed after such repetitive sequences have been removed from the DNA. We have applied our newly designed subtraction strategy to microdissected chromosomes in generating whole chromosome painting (e.g., chromosome 4), chromosome arm (e.g., 1q and 15q), and band (e.g., 3q26) specific probes, and we have demonstrated its utility using flow-sorted chromosome. FISH analyses using these probes show consistent strong signals with no cross-hybridization, and optimal hybridization results can be obtained relatively quickly.

Characterization of a human alphoid satellite DNA sequence: Potential use in assessing genetic diversity in Indian populations

Sequence analysis of a human repetitive DNA sequence (pTRF5.6) revealed considerable homology (76%) to the alphoid consensus sequence. Genomic blots of StuI-digested human DNA, hybridized to pTRF5.6, generated a ladder of bands with each band corresponding to oligodeoxyribonucleotide of an approx. 170-bp repeat, indicating a tandemly arrayed organization of this repeat element within the genome. Genomic hybridization analyses of unrelated individuals belonging to various geographical regions of India, using this alphoid satellite probe, revealed polymorphic bands ranging between 2 and 9 kb. Along with an individual-specific band pattern, several isomorphic bands below 2 kb were also evident. There was very little of genetic variability between populations, suggestive of low polymorphism at the inter-population level. Our result suggest that alphoid satellite sequence probe can be used in assessing the genetic diversity of various ethnic groups/populations belonging to different geographical regions.

The Human Telomere

An ultimate goal of human genetics is the generation of a complete physical and ''functional'' map of the human genome. Twenty-five percent of human DNA, however, consists of repetitive DNA sequences. These repetitive DNA sequences are thought to arise by many mechanisms, from direct sequence amplification by the unequal recombination of homologous DNA regions to the reverse flow of genetic information. A general outline of the chromosomal organization of these repetitive sequences will be discussed. Our working hypothesis is that certain classes of human repetitive DNA sequences ''encode'' the information necessary for defining long-range genomic structure. Evidence will be presented that the first goal of this research, the identification and cloning of the human telomere, has been achieved. A human repetitive DNA library was constructed from randomly sheared, reassociated, and oligo(G/center dot/C)-tailed DNA, a method that minimizes the potential loss of sequences devoid of a given restriction enzyme site. Sequences too large to clone efficiently in cosmid or /lambda/ vectors, such as centromeric repeats, or telomeric sequences with an end incompatible for cloning, should be present in this library. In order to isolate highly conserved repetitive DNA sequences, this library was screened with radiolabeled hamster Cot50 repetitive DNA. Two clones, more » containing tandem arrays of the sequence (TTAGGG), were isolated by this method. 30 refs., 1 fig., 2 tabs. « less

Molecular and functional analysis of tumor-suppressor genes by transfection.

The transformed and tumorigenic phenotype of H-ras transfected rat FE-8 cells can be suppressed by cell fusion with normal rat embryo fibroblasts. Transfection into FE-8 cells of DNA prepared from normal human placenta followed by selective elimination of tumorigenic transfected cell clones resulted in the isolation of phenotypically normal revertants. These cells exhibited a fibroblastlike, normal morphology; were anchorage-dependent; and were unable to proliferate in medium with reduced serum concentrations. Their tumorigenicity was also reduced. The suppressed phenotype has been transferred in a second transfection cycle. Human repetitive DNA sequences were detected in secondary transfectant DNA. A putative human suppressor gene, designated NTS-1, has been molecularly cloned. Reintroduction of cloned NTS-1 sequences into FE-8 cells resulted in suppression of the neoplastic phenotype in spite of a high ras expression.

A nuclear protein associated with human cancer cells binds preferentially to a human repetitive DNA sequence.

A protein (Rp66) of 66 kDa was shown by DNA-binding protein blot assay to bind to a human repetitive DNA sequence (low-repeat sequence; LRS) in each of 10 transformed human cell lines examined. This protein-DNA interaction was not observed in 11 normal human cell cultures or in the Chinese hamster cell line CHO-K1. Gel retardation assay confirmed the specificity of the protein-DNA binding between Rp66 and LRS. In a histiocytic lymphoma human cell line, U937, that can be induced to differentiate in the presence of phorbol ester, this binding disappeared after cell differentiation. These together with other results cited suggest a regulatory role for these repetitive sequences in the human genome, with particular application to cancer.

Cytoplasmic localization of human repetitive DNA revealed by in situ hybridization

Previously, we showed that a human repetitive DNA sequence ( Sau3A family) belonging to a satellite DNA is unstable and constantly excised from the chromosomes ( R. Kiyama, H. Matsui, and M. Oishi, 1986, Proc. Natl. Acad. Sci. USA 83, 4665). The unusual property of the repetitive DNA, along with another repetitive DNA ( Alu sequence), was further investigated by in situ hybridization in several different human cells including HeLa, bone marrow, and peripheral blood cells. We found that the excised repetitive DNA sequences are localized not only in nuclei, but also in cytoplasm. These results have confirmed the instability of these DNA sequences in the chromosomes and further suggest that the a satellite DNA and the Alu sequence which were excised from the chromosomes are released from nuclei to cytoplasm.

Development of neoplastic phenotype following transfection of HNF cells with sarcoma DNA.

DNA isolated from chondrosarcoma cells effectively transformed NIH-3T3 cells and human foreskin fibroblasts. The transfected NIH-3T3 cells, directly implanted three or four passages later, formed progressively growing tumors (greater than or equal to 2.0 cm in diameter) subcutaneously in nude mice. No metastasis was evident upon pathological examination of the tumor bearing mice. Transfected human foreskin fibroblasts that exhibited anchorage independent growth formed only small tumors in nude mice (less than 0.6 cm in diameter). The transfected human cells which exhibited anchorage independent growth reacted with the monoclonal antibody 345.134S, specific for an epitope expressed by human sarcoma cells. The transfected NIH-3T3 cells did not exhibit reactivity with the same monoclonal antibody. Southern blot analysis of the DNA prepared from the transfected NIH-3T3 cells, that developed as a progressively growing tumor in a nude mouse, revealed the presence of human repetitive DNA sequences.

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.

Amplification of Genes Encoding Human Myeloid Membrane Antigens After DNA-Mediated Gene Transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH-3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.

Molecular cloning of Ancient Egyptian mummy DNA

Artificial mummification was practised in Egypt from approximately 2600 BC until the fourth century AD. Because of the dry Egyptian climate, however, there are also many natural mummies preserved from earlier as well as later times. To elucidate whether this unique source of ancient human remains can be used for molecular genetic analyses, 23 mummies were investigated for DNA content. One 2,400-yr-old mummy of a child was found to contain DNA that could be molecularly cloned in a plasmid vector. I report here that one such clone contains two members of the Alu family of human repetitive DNA sequences, as detected by DNA hybridizations and nucleotide sequencing. These analyses show that substantial pieces of mummy DNA (3.4 kilobases) can be cloned and that the DNA fragments seem to contain little or no modifications introduced postmortem.

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [ 3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [ 3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.

Characterization of reiterated human DNA with respect to mammalian X chromosome homology.

Recombinants containing human repetitive DNA sequences were identified by dot hybridization and classified with respect to presence on the X chromosome and homology to mouse DNA. Using genomic probes that differ in number of X chromosomes, we observed extensive homology between human autosomal and X sequences. Hybridization to genomic probes that differ in species of origin indicate that these reiterated sequences have diverged between mouse and man. Eleven recombinants, each containing a different reiterated sequence(s), were hybridized in situ to metaphase chromosomes of mouse and man. These studies indicate that reiterated DNA which is homologous to the human X chromosome is more similar to DNA of human autosomes than to any murine chromosome. Therefore, it seems that reiterated DNA sequences on the human X chromosome have diverged as much during mammalian evolution as sequences on human autosomes. Moreover, the extensive modification of the original mammalian X has not interferred with the X inactivation process.