Linkage in human heterochromatin between highly divergent Sau3A repeats and a new family of repeated DNA sequences (HaeIII family)

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp] were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a "new" family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.     

Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

Chromosomal location by in situ hybridization of the human Sau3A family of DNA repeats

Summary The Sau3A family is a human, clustered, highly repetitive, GC-rich DNA family. In situ hybridization studies with a plasmid carrying a Sau3A monomer as a probe have shown that Sau3A sequences are preferentially concentrated in the heterochromatic regions of human acrocentric chromosomes (D and G groups, both in pericentromeric regions and in cytological satellites) and in pericentromeric heterochromatin of chromosome 1. The same chromosomal locations were observed by using as probes two recombinant phages which carry Sau3A-positive genomic sectors. The two sectors differfor the relative proportions of monomer and multiples of Sau3A repeats, which show different extents of homology to the cloned monomer, and for the presence, in one of the two, of a samll amount of an unrelated repeat (alphoid DNA). The similarity of the results obtained with the three probes suggests that heterogeneous Sau3A repeats share the same chromosomal localizations and that the two analyzed genomic sectors may not contain significant amounts of repetitive DNAs other than the Sau3A family. A comparison between the chromosomal locations of Sau3A and EcoRI families of repeats has confirmed that each family is characterized by specific chromosomal locations and that single heterochromatic regions may contain both.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

Human Sau3A repeated DNA is enriched in small polydisperse circular DNA from normal lymphocytes

Representatives of the Sau3A family of short human repeated sequences [Meneveri et al., J. Mol. Biol. 186 (1985) 483-489] have been isolated from the small polydisperse circular DNA (spcDNA) of peripheral human lymphocytes. The prototype repeat is a 72-bp element which is at least partially tandemly repeated in spcDNA and human genomic DNA. In comparison with three major families of human repeated DNA, the Sau3A repeats are enriched in spcDNA. The function of spcDNA in normal and transformed eukaryotic cells is not understood and most studies have attempted to resolve this problem by molecular analysis of circular DNA isolated from cells in culture [see Rush and Misra, Plasmid 14 (1985) 177-191 for references]. We have studied the spcDNA present in normal uncultured human lymphocytes and present data pointing to the selective accumulation of the Sau3A family of repeated DNA within this population. The sequences of twelve of these repeats, the consensus sequence for this family and the sequence of a genomic repeat, are presented.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

A family of long reiterated DNA sequences, one copy of which is next to the human beta globin gene.

An unusually long repeated DNA sequence was identified in cloned DNA, three kb 3' to the human beta-globin gene. Other members of this repeated sequence family were isolated from a human genomic DNA library and characterized by Southern blotting techniques, electron microscopy, and solution hybridization. The copy located next to the beta-globin gene was found to be 6.4 +/- 0.2 kb long and continuous over that length. This repeated sequence family comprises about 1% of the human genome and contains 3000-4800 copies of moderate sequence divergence which are interspersed with other less-highly repeated DNA. The 6.4 kb repeated unit does not appear to be composed of any smaller tandemly repeated subunits, nor is it expressed at a high level in bone marrow cell RNA.     

Structure of the pericentric long arm region of the human Y chromosome.

We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome. The structures of one cosmid and three yeast artificial chromosome clones were determined. The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families. A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.     

The 724 family of DNA sequences is interspersed about the pericentromeric regions of human acrocentric chromosomes

We describe the organization of the complex, interspersed 724 family of DNA sequences that is distributed in multiple copies about the pericentromeric region of human acrocentric chromosomes. 724 family members were isolated using an efficient recombination-based assay for nucleotide sequence homology to screen a human genomic library. Eight related but distinct 724 family members were isolated that hybridized to a total of 20 different human-genomic EcoRI DNA fragments spanning 100,000 base pairs. In contrast with tandemly clustered satellite and ribosomal DNA sequences also located on the short arms of human acrocentric chromosomes, 724 family members are interspersed. No evidence for local interspersion or homology between 724 family members and ribosomal or satellite DNA sequences was found. Juxtaposition of the complex 724 family to the nucleolus organizer region was a recent event in primate evolution. The unique organization of 724 family members on each of the five human acrocentric chromosomes indicates that the 724 family continues to evolve within the human karyotype.     

A group of repetitive human DNA families that is characterized by extrachromosomal oligomers and restriction-fragment length polymorphism

We have recently reported a novel human repetitive DNA (Sau3A family) that exists both in the chromosomes and in the extrachromosomal fraction. Several more clones that hybridized with the Sau3A family were isolated from the extrachromosomal fraction of HeLa cells. Use of these clones as probes has revealed that at least four different types of oligomeric forms of DNA are present in the extrachromosomal fraction. The oligomers consist of one, two, five or 12 subunits of basic 170 base-pair unit DNA, or superimposed forms of two of them. Nucleotide sequencing of these clones indicated that the clones have 70 to 90% sequence homology with human alphoid satellite DNA. These DNA sequences are present also in the chromosomes, as tandemly repeated DNA sequences, and exhibit a considerable degree of restriction-fragment length polymorphism. These results, taken together with the previous findings on Sau3A family DNA, suggest that there is a group of recombination-prone repetitive DNA families in human chromosomes.     

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.     

Genomic and cDNA sequence tags of the hyperthermophilic archaeon Pyrobaculum aerophilum.

The hyperthermophilic archaeum, Pyrobaculum aerophilum, grows optimally at 100 degrees C with a doubling time of 180 min. It is a member of the phylogenetically ancient Thermoproteales order, but differs significantly from all other members by its facultatively aerobic metabolism. Due to its simple cultivation requirements and its nearly 100% plating efficiency, it was chosen as a model organism for studying the genome organization of hyperthermophilic ancient archaea. By a G+C content of the DNA of 52 mol%, sequence analysis was easily possible. At least some of the mRNA of P. aerophilum carried poly-A tails facilitating the construction of a cDNA library. 245 sequence tags of a poly-A primed cDNA library and 55 sequence tags from a 1-2 kb Sau3AI-fragment containing genomic library were analyzed and the corresponding amino acid sequences compared with protein sequences from databases. Fourteen percent of the cDNA and >9% of genomic DNA sequence tags revealed significant similarities to proteins in the databases. Matches were obtained to proteins from archaeal, bacterial and eukaryal sources. Some sequences showed greatest similarity to eukaryal rather than to bacterial versions of proteins, other matches were found to proteins which had previously only been found in eukaryotes.     

Cloning of a yolk protein gene family from Caenorhabditis elegans

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA-dependent. The rhsA locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhsA are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhsA or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed "transductional walking", by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.     

Yeast thioredoxin genes

Based on the conserved protein sequence of thioredoxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA. A 34-base pair (bp) sequence around the active site of yeast thioredoxin was obtained from the polymerase chain reaction product. This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes. Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast genomic library. Two Sau3A1 fragments, 825 and 2045 bp, respectively, from two different clones were cloned into pUC13. Sequence analysis of these fragments gave two different open reading frames without introns. The 825-bp Sau3A1 fragment encodes a 103-amino acid residue protein named thioredoxin I. The 2045-bp Sau3A1 fragment contains a sequence encoding thioredoxin II which has 102 amino acid residues. This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source. Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys. Thioredoxins I and II show 78% amino acid sequence identity. They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chloroplast thioredoxins.     

Organization of the gene encoding the mouse T-cell-specific serine proteinase "granzyme A".

The mouse serine protease granzyme A is a member of a closely related family of T-cell-associated proteolytic enzymes, designated granzymes A-G. Previous studies have indicated that granzymes A and B are involved in various T-cell-mediated processes. Here we report the genomic organization of the granzyme A gene. We have cloned a 15-kb DNA fragment from a genomic library of a cloned CD8+ T-cell line and sequenced the exon-intron boundaries. The gene consists of five exons, and its genomic organization is very similar to that described for granzymes B, C, and F. In addition, we have sequenced 1.4 kb of the 5'-region and 1.1 kb of the 3'-region flanking the granzyme A gene. Putative promoter and enhancer elements were identified by sequence comparison with known consensus sequences. Some of these regulatory elements seem to be associated exclusively with granzyme A, whereas others are shared by members of the granzyme family.