Highly repetitive DNA sequence in parthenogeneticArtemia

Summary The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species.Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy).Digestion of genomic DNA of the parthenogeneticArtemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogeneticArtemia populations and compared with an Alu I repetitive fragment previously identified inArtemia franciscana.     

The selective digestion of polytene and mitotic chromosomes of Drosophila melanogaster by the Alu I and Hae III restriction endonucleases

Polytene and mitotic chromosomes of Drosophila melanogaster were treated with either Alu I or Hae III restriction endonucleases. Subsequent staining with the DNA-specific fluorochrome ethidium bromide showed that these enzymes are capable of selectively digesting chromosomal DNA in fixed cytological preparations, as previously shown in mammalian metaphase chromosomes. Alu I or Hae III digestion made possible the localization in situ of some highly repetitive DNAs in both polytene and mitotic chromosomes, while only Alu I permitted the localization of the 5S RNA genes on the polytene chromosomes of D. melanogaster.     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.     

Preparation of centromeric heterochromatin by restriction endonuclease digestion of mouse L929 cells

When L929 cells in metaphase are digested with either Eco RI or Alu I, chromatin containing about 85% of the DNA is released. DNA from the Alu I- and Eco RI-resistant chromatin is enriched 6.8- and 3.7-fold, respectively, in satellite sequences. Analysis by electron microscopy of these digests reveals the existence of structures containing condensed heterochromatin and kinetochores. When these preparations are incubated with anticentromere serum from a human CREST scleroderma patient and then with rhodamine-conjugated antihuman IgG, fluorescence appears in the form of paired dots, the same pattern found in whole metaphase chromosomes. The fluorescent staining pattern, the electron microscopy, and the enrichment of satellite DNA sequences together support the conclusion that the Eco RI- and Alu I-resistant structures contain centromeres. We anticipate that these preparations will be useful in studies of the interactions between centromeric heterochromatin, kinetochores, and microtubules.     

Genetic Mapping of a Possible New Alcohol Dehydrogenase Sequence to Mouse Chromosome 3 at the Adh-1/Adh-3 Complex

Southern blot analysis of mouse genomic DNA reveals two Eco RI fragments which faintly hybridize to mouse Adh-1 cDNA and are not part of the Adh-1 gene. These fragments were isolated from agarose gels, cloned, and characterized. Sequence analysis of the 2.1-kb Eco RI fragment suggests that it is likely a pseudogene since it does not contain a long open reading frame. However, the 2.0-kb Eco RI fragment contains a coding sequence with a long open reading frame which corresponds to exon 6 of the mouse Adh-1 gene. Comparison of the coding sequence with other known ADHs suggests that the sequence has diverged sufficiently from any currently known class of ADH to be a possible distinct class. Further confirmation awaits analysis of currently available genomic clones. Using these sequences as probe, restriction fragment length polymorphisms were identified for each sequence between C57Bl/6J and DBA/2J inbred mouse strains. The strain distribution pattern for these allelic differences was determined among the B × D recombinant inbred strains. This analysis revealed that the 2.1-kb Eco RI sequence is located on chromosome 3 but at a distance from the Adh-1/Adh-3 complex as previously reported. However, the new polymorphism identified in the 2.0-kb Eco RI fragment enabled this sequence to be mapped at the Adh-1/Adh-3 complex.     

The banding pattern produced by restriction endonucleases in mouse chromosomes

The metaphase chromosomes of mouse have been treated with the restriction endonucleases Alu I, Mbo I, Hae III and Eco RII and subsequently stained with the DNA-specific dye Ethidium bromide. A striking correspondence between previous biochemical findings and our cytological results has been noticed with Alu I, Mbo I and Hae III, which were capable of digesting all but the DNA localized at the centric constitutive heterochromatic areas. Digestion with Eco RII, on the contrary, resulted in cytological data which apparently did not fit in with the biochemical results previously obtained by digesting the DNA of this species with the same enzyme. It is postulated that factors such as chromatin organization, in addition to DNA base sequence, can determine the results we report.     

Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions.

Bacterial restriction endonucleases containing the dinucleotide CpG in their cleavage sequences were used to compare the methylation patterns of primarily repeated DNA sequences in (1) bovine somatic cell native DNAs vs bovine sperm cell native DNA and (2) native vs renatured bovine liver and sperm cell DNAs. The restriction patterns of sperm native DNA differ markedly from those of somatic cell native DNAs when using Hpa II, Hha I, and Ava I but not when using the enzymes Eco RI and Msp I. Digestion patterns of germ cell renatured DNA differed significantly from those of germ cell native DNA when using Hpa II but not when using Msp I or Eco RI. The results may not be due to artifacts of renaturation of the DNAs. The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated. The data also suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Recent amplification of an alpha satellite DNA in humans.

A repeat sequence 682 base pairs (bp) long produced by cleavage of human DNA with Xba I restriction enzyme is composed of four tandemly arranged subunits with lengths of 171, 170, 171, and 170 bp each. The sequence organization of the 682 bp Xba I repeat bears a striking resemblance to other complex satellite DNAs of primates, including the Eco RI human alpha satellite family which also occurs as a 170 bp repeat. The Eco RI tetramer and the 682 bp Xba I repeat show a sequence divergence of 21%. The 682 bp Xba I repeat sequence is restricted to humans and is only distantly related to the previously reported 340 bp Xba human repeated DNA sequence. These finding are consistent with the concept of occasional amplifications of members or groups of members of alpha satellite DNA during human evolution. Amplifications apparently occurred after humans, apes and gibbons diverged from Old World monkeys (Eco RI satellite), after humans and apes diverged from gibbons (340 bp Xba I satellite) and after humans diverged from the great apes (682 bp Xba I satellite).     

Interspersed repetitive and tandemly repetitive sequences are differentially represented in extrachromosomal covalently closed circular DNA of human diploid fibroblasts.

Extrachromosomal covalently closed circular DNA (cccDNA) was isolated from human diploid fibroblasts by alkaline denaturation/renaturation and CsCl-ethidium bromide isopycnic centrifugation. Probing across these gradient fractions showed a higher proportion of cccDNA sequences homologous to the interspersed highly repetitive Alu I and Kpn I sequences than to the human tandemly-repetitive Eco RI (alphoid) DNA. Cloning of these cccDNAs was then carried out following digestion with restriction endonucleases Hind III, Bam HI or Pst I, and ligation into plasmid pBR322. Many isolated recombinant clones were unstable as seen by a high rate of loss over four cycles of antibiotic selection, and frequent plasmid modifications including deletions adjoining the site of insertion. Of 107 cloned sequences which appeared relatively stable, i.e., survived four cycles of antibiotic selection without incurring detectable deletions, 28% and 11% showed homology to Alu I and Kpn I families, respectively, while 4% contained sequences homologous to both. In contrast, less than one percent hybridized to probes for tandemly-repetitive sequences, Eco RI and Satellite III. The average insert size of cloned cccDNA derived from human fibroblasts, 2.52 Kbp, was larger than previously reported for similar clones derived from genetically less stable permanent lines, which may reflect differences in the process of cccDNA generation.     

Methylation of Eco RI (GAm'ATTC) sequences of bacterial DNA

The methylation of Eco RI (GAm6 ATTC) sequences of DNA of bacteria related to the main branches of their phylogenetic dendrogramme, was studied. It was found that methylation of Eco RI sites is observed in bacteria Caulobacter and in Thermus aquaticus. This finding can be substantiated by the resistance of these DNAs to Eco RI restrictase as well as by the fact that Bam HI fragments of these DNAs cloned within the composition of the vector plasmid pUC8 in E. coli cells contain GAATTC sites.     

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

Distribution and frequency of a polymorphicAlu insertion at the plasminogen activator locus in humans

We have investigated the frequency distribution, across a broad range of geographically dispersed populations, of alleles of the polymorphic Alu insertion that occurs within the 8th intron of the tissue plasminogen activator gene (PLAT). This Alu is a member of a recently derived subfamily of Alu elements that has been expanding during human evolution and continues to be transpositionally active. We used a “population tube” approach to screen 10 chromosomes from each of 19 human populations for presence or absence of this Alu in the PLAT locus and found that all tested populations are dimorphic for presence/absence of this insertion. We show that the previously published EcoRI, HincII, PstI, TaqI, and XmnI polymorphisms at the PLAT locus all result from insertion of this Alu and we use both restriction fragment length polymorphism and polymerase chain reaction analysis to examine the frequency of Alu(+) and Alu(–) alleles in a sample of 1003 individuals from 27 human populations and in 38 nonhuman primates. Nonhuman primates are monomorphic for the Alu(–) allele. Human populations differ substantially in allele frequency, and in several populations both alleles are common. Our results date the insertion event prior to the spread and diversification of modern humans. Received: 10 July 1995 / Revised: 17 November 1995     

The Alu I-induced bands in metaphase chromosomes of orangutan (Pongo pygmaeus)

Summary Restriction endonucleases have been recently proved to be active on fixed chromosomes, thus they are useful in chromatin structure studies. Within this class of enzymes, Alu I is able to detect the presence and localization of highly repetitive DNA sequences in human and in other mammalian and dipteran species. In this paper the pattern obtained on fixed metaphase chromosomes of orangutan (Pongo pygmaeus) by Alu I digestion and Giemsa staining is shown. The results are discussed in the light of the distribution, in this species, of the I–IV human satellite DNAs. It is also suggested that in Pongo some highly repetitive sequences, different from the major human satellites, are present.     

Restriction endonuclease analysis of human globin genes in cellular DNA

Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hinc II, Bam HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were transferred to nitro-cellulose filters and identified by hybridization to [³²P] cDNA for total adult globin mRNA. The α-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous α-thalassemia, a genetic disorder due to deletion of α-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated α-globin gene loci.     

Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli β-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and β-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5·10⁴ molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.     

Mobility of short interspersed repeats within the chimpanzee lineage

The PV subfamily of Alu repeats in human DNA is largely composed of recently inserted members. Here we document additional members of the PV subfamily that are found in chimpanzee but not in the orthologous loci of human and gorilla, confirming the relatively recent and independent expansion of this Alu subfamily in the chimpanzee lineage. As further evidence for the youth of this Alu subfamily, one PV Alu repeat is specific to Pan troglodytes, whereas others are present in Pan paniscus as well. The A-rich tails of these Alu repeats have different lengths in Pan paniscus and Pan troglodytes. The dimorphisms caused by the presence and absence of PV Alu repeats and the length polymorphisms attributed to their A-rich tails should provide valuable genetic markers for molecular-based studies of chimpanzee relationships. The existence of lineage-specific Alu repeats is a major sequence difference between human and chimpanzee DNAs. Correspondence to: C.W. Schmid     

Preparation and properties of insolubilized restriction endonucleases.

Type II restriction endonucleases Bam HI and Eco RI were covalently coupled to Sepharose. These insolubilized enzymes generated fragment patterns for several viral DNAs identical to those produced by the respective free enzymes. Conditions for optimal activity were similar for both bound and unbound forms of the enzymes. Insolubilization improved thermal stability of Bam HI and Eco RI. The bound enzyme can be recovered from reaction mixtures and reused several times. Upon storage at 4 degrees C, coupled endonucleases remained stable for several months.     

Isolation of deletion and substitution mutants of adenovirus type 5

The infectivity of adenovirus type 5 DNA can be increased to about 5 x 10³ plaque-forming units per μg DNA if the DNA is isolated as a DNA-protein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (1–76 map units), fragment C (76–83 map units) and fragment B (10–83 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin.