Dose-independent uniform distribution of UV-induced repair replicated DNA among DNA sequences of different reiteration frequencies in cultured human fibroblasts

The distribution of UV-induced repair replicated DNA sequences among highly reiterated, moderately reiterated, and single copy human DNA has been investigated by molecular reassociation at various UV doses. Results indicate that repair replicated DNA patches are uniformly distributed within highly repeated, moderately repeated, and single copy DNA sequences at every dose of UV radiation.     

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.     

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.     

An electron microscope study of the DNA sequence organization of the human genome

The arrangements of inverted-repeated and repeated DNA sequences in the human genome have been investigated by an electron microscope method. The arrangement of the interspersed repeated DNA sequences is found to be similar to the corresponding arrangement found in Xenopus. This arrangement consists of 300-nucleotide-long repeated DNA sequences interspersed with roughly gene-size single-copy DNA sequences. The inverted-repeated sequences are also 300 nucleotides in length and are interspersed with the other DNA sequence classes.Most inverted-repeated sequences (64%) are spaced by another sequence which is recognized by electron microscopy as a single-stranded loop in a hairpin structure. The average length of this spacer loop is 1.6 kilobases. Although some pairs of inverted-repeated sequences are clustered, most seem to be randomly distributed throughout the genome. The average distance separating two pairs of inverted-repeated sequences is 10 to 20 kilobases. The interspersed repeated sequences and inverted-repeated sequences are arranged simultaneously in a portion of the human genome resulting in an interspersion of all three sequence classes.     

A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

The evolution of a family of short interspersed repeats in primate DNA

Summary Human DNA contains 300 nucleotide interspersed repeated sequences which mostly belong to a single family of sequences called the Alu family. This work examines the evolution of this family of sequences in primates. Bonnet monkey (Macaque radiata) DNA contains a predominant family of 300 nucleotide repeats which has nearly the same restriction map as the human Alu family and which hybridizes to human Alu family repeats under Southern blotting conditions. Prosimian (Galago crassicaudatus pangeniesis) DNA also contains a prominent group of 300 nucleotide long repeated sequences which does not have the same restriction sites as the human Alu family but which does hybridize to the human Alu family under reduced stringency conditions.     

Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes

The analysis of a recombinant human Y-enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with one single copy or slightly repeated Y-specific DNA sequence, Y-specific repeated sequences of various restriction fragment lengths, Y-chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, Y-specific DNA sequences in addition to multiple X and/or autosomal sequences, or Y-specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46,XY patients with gonadal dysgenesis and small deletions of the Y short arm.     

Cloning of a repeat sequence from human DNA which contains a BamHI site

A human 1.5 kilobase BamHI repeated DNA fragment has been cloned from a genomic library and subcloned in pBR322. It is part of the human homogeneous main-band DNA, it has properties similar to those of long interspersed repetitive sequences (LINES), and differs from the families of human repeated DNA already described.     

Molecular hybridization to meiotic chromosomes in man reveals sequence arrangement on the no. 9 chromosome and provides clues to the nature of "parameres"

In situ hybridization of male human meiotic material has been used to elucidate the molecular organization of the centromeric region of human chromosome 9. The use of two cloned DNA sequences has shown that the centromere and the secondary constriction of this chromosome contain two separate repeated DNA families. The secondary constriction organizes into "paramere" bodies during pachytene. The individual parameres are comprised of one family of repeated DNA sequences.     

A member of a new repeated sequence family which is conserved throughout eucaryotic evolution is found between the human delta and beta globin genes

A new class of human interspersed repeated sequences distinct from the AluI family was found by screening a human gene library with a mouse ribosomal gene non-transcribed spacer probe (rDNA NTS). A member of this sequence family was localized to a 251 bp segment between the human delta and beta globin genes: a region previously judged to be devoid of repeated DNA. The complete nucleotide sequence of this segment revealed a tandem block of 17 TG dinucleotides, a feature hypothesized by others to be a recombination hot spot responsible for gene conversion in the gamma globin locus region. When the genomes of Xenopus, pigeon, slime mold and yeast were examined, reiterated sequences homologous to both the mouse rDNA NTS and human globin repeat were found in every case. The discovery of this extraordinarily conserved repeated sequence family appears to have depended upon not using salmon sperm DNA during hybridization. The use of eucaryotic carrier DNA may bias the search for repeated sequences against any which may be highly conserved during eucaryotic evolution.     

A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.     

A new approach to cloning of tandem repetitive DNA sequences

A new approach to screening of the repeated human DNA sequences tandemly arranged in the genome is described. Efficiency of the developed approach for search of tandemly arranged DNA sequences is corroborated by the obtained experimental data.     

Regulated expression of human growth hormone genes in mouse cells

We have asked whether there are sequences around the human growth hormone gene that render this gene responsive to induction by glucocorticoid hormones. Recombinant clones encoding human growth hormone were introduced into the chromosome of murine fibroblasts by cotransformation. Exposure of cotransformants to glucocorticoids results in a three to five fold induction of human growth hormone mRNA and a similar induction in secreted human growth hormone protein. The DNA sequences required for induction reside within 500 nucleotides of 5′-flanking DNA. Fusion of this segment of 5′-flanking DNA to the structural gene sequences of a hormone-insensitive gene, such as thymidine kinase, now renders this gene responsive to glucocorticoid induction.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.     

The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases

Cloned murine mammary tumor virus (MuMTV) sequences allowed us to search for murine mammary tumor virus related sequences in the DNA of surgically removed human breast tumors. Out of 28 tumors so far examined two were found to contain an Eco RI DNA fragment homologous to the long terminal repeat-group antigen (LTR-Gag) and the Envelope (Env) sequences of MuMTV. We have taken the lymphocytes of these patients and cultured them. Rapid growth of lymphocytes, mostly of T origin, occurred in the presence of T cell growth factor (TCGF). Whereas DNA extracted from fresh lymphocytes is negative, that extracted from the 3-day cultured lymphocytes showed MuMTV related sequences. Long term cultures of T cells and a similar culture derived from a healthy person donor were negative at all stages. DNA extracted from the Ebstein Barr Virus-transformed B cells of the patient does not contain the MuMTV related sequences.