KpnI families of long, interspersed repetitive DNAs in human and other primate genomes.

KpnI restriction of DNAs from all anthropoid primates studied releases a conspicuous series of segments representing families of long, interspersed repetitive DNAs termed here the KpnI 1.2, 1.5, 1.8 and 1.9 kb families. Human KpnI 1.2 to 1.9 kb segments representative of these families were isolated and separately cloned in the KpnI site of a plasmid pBK5, specially constructed for this purpose. The KpnI clones did not cross-hybridize with cloned, primate alphoid sequences, suggesting that the KpnI families represent sequences separate and distinct from the alphoid DNAs. Secondary restriction analyses of cloned KpnI segments demonstrated microheterogeneity among individual members within the same KpnI family. Autoradiograms of capuchin monkey, AGM and human DNA cleaved with HaeIII, AluI or RsaI and hybridized to various cloned human KpnI sequences demonstrated a remarkable conservatism and relative simplicity in the organization of the KpnI families in the genomes of these widely divergent primates. The KpnI 1.2 kb and 1.5 kb families occur in high frequency (15%) among all plaques in two recombinant human genome libraries. Evidence is presented suggesting that the bulk of the KpnI families occur in the genome as clusters or congeries of higher molecular weight segments (greater than 2 kb) containing sequences homologous to the low molecular weight segments (1.2 to 1.9 kb).     

Nuclear and mitochondrial DNA have sequence homology with a chloroplast gene

Summary A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P₇₀₀ chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.     

Insertion of a long KpnI family member within a mitochondrial-DNA-like sequence present in the human nuclear genome

Structural analysis of a phage lambda Charon 4A clone carrying one of the human nuclear mitochondrial(mut)-DNA-like sequences revealed that a KpnI-family member (KpnI 5.5-kb DNA) is inserted within this sequence. The inserted KpnI 5.5-kb DNA contains several possible polyadenylation signal sequences followed by an A-rich sequence at its 3' end and is flanked by perfect 13-bp direct repeats of the duplicated mtDNA-like sequences. These structures strongly suggest that the KpnI 5.5-kb DNA is a mobile element. Comparison of the 5' terminal sequences of the KpnI 5.5-kb DNA and four other long KpnI-family DNAs so far examined, using the predicted general promoter sequence for eukaryotic tRNAs, indicates that they contain the consensus sequences for the split internal RNA polymerase III control region.     

A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.     

Sequence analysis of a KpnI family member near the 3' end of human beta-globin gene.

We determined the complete nucleotide sequence (6125 bp) of a full-length member of human KpnI family, designated T beta G41, which is located about 3 kb downstream from the beta-globin gene. Comparison of the sequence with the KpnI family sequence compiled by Singer revealed that a new 131 bp sequence is present in the T beta G41. Hybridization analyses showed that a few thousand of human KpnI family members are carrying this additional sequence. Computer search of DNA databases for T beta G41-homologous sequence showed that some T beta G41-homologous sequences were closely associated with pseudogenes. The T beta G41 sequence also showed significant sequence homology with ChBlym-1, a transferrin-like transforming gene of chicken. Furthermore, an amino acid sequence deduced from the T beta G41 nucleotide sequence revealed a relatively-high homology to those of human transferrin and lactotransferrin.     

The effect of E. coli host strain on the consensus sequence of regions of the human L1 transposon.

We have used highly methylation tolerant host strains to clone hyper- and hypo-methylated genomic elements from different regions of the same family of long interspersed repetitive elements from human DNA, specifically the 1.8 kilobase (kb) and 1.2kb KpnI fragments from members of the L1 family of transposable elements in which respectively some 18% and 2.7% of cytosines are methylated in vivo in human spleen DNA. The consensus of the DNA sequences of the ends of 13 clones from the hypomethylated region of human L1 agreed exactly with the consensus derived previously from clones made using conventional host strains. However the sequences of 18 of our clones from the 5' end of the hypermethylated region differed significantly from the sequences of clones made using conventional hosts (P less than 0.0001). The 5' region of the 1.8kb L1 region is a CpG island which, in human somatic tissue, appears to be maintained in a highly methylated state, including methylation at sites other than CpG dinucleotides. The consensus sequence of this region also has features suggestive of a previously unrecognized open reading frame.     

The organization of two related subfamilies of a human tandemly repeated DNA is chromosome specific

Summary Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1 kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).     

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.     

The association of the interspersed repetitive KpnI sequences with the nuclear matrix

The KpnI sequences constitute the dominant, long, interspersed repetitive DNA families in primate genomes. These families contain related, but nonidentical sequence subsets, some of which border functional gene domains and are transcribed into RNA. To test whether these sequences perform an organizational function in the nucleus, their association with the nuclear matrix has been examined in African green monkey cells. DNase I treatment depleted the residual matrix of most of the KpnI 1.2- and 1.5-kilobase pair family sequences although significant amounts of each family remained in the loop attachment DNA fragments. Hybridization analysis of the KpnI and RsaI cleavage patterns of matrix loop attachment DNA indicate that some sequence subsets of these KpnI families are relatively less depleted than others. The nuclear matrix association of subpopulations of KpnI 1.2- and 1.5-kilobase pair families was also shown by metrizamide gradient centrifugation of nuclear matrix complexes cleaved by KpnI endonuclease. The gradients demonstrate that some KpnI segments are differentially associated with nuclear matrix proteins. Moreover, the procedures permit the preparative isolation and purification of the DNA-protein complexes containing these KpnI 1.2- and 1.5-kilobase pair sequence families. Speculations on the relationship between the matrix association of these KpnI family sequences and their possible roles in gene organization and expression are presented and discussed.     

Toward a molecular paleontology of primate genomes

KpnI restriction of anthropoid primate DNAs, from a New World monkey to man, releases a series of segments that are remarkable among all of the alphoid DNAs in the constancy of their relative amounts in the various primate genomes, in their long-range organization, and in their internal sequence structure. These segments are labeled the KpnI A, B, C and D segments. Cross-hybridization analysis by Southern filter-transfer hybridization indicates that the KpnI segments represent separate and distinct families of alphoid DNAs. These families are termed the KpnI A, B, C and D families of alphoid sequences, of which only the KpnI A and B families were studied in detail here. - Evidence is presented suggesting that the KpnI segments do not exist as long, tandemly repeated sequences in the primate genome: rather, they may occur interspersed among other, perhaps nonalphoid sequences. From the stained gel patterns and from Southern filter-transfer hybridization experiments, the KpnI families appear to be absent from the genomes of the two prosimians studied - the galago and the black lemur. The KpnI A and B families are found among all of the anthropoid primates, including the New World capuchin monkey. The KpnI C family was detected in the genomes of the Old World anthropoid primates whereas the KpnI D family was detected only among the great apes and man. - The results are in accord with the observation (Musich et al., 1980) that with the continued evolutionary development of the primate Order, there has been a parallel trend toward an increased number and variety of alphoid DNA sequences. The properties of the KpnI families suggest that these sequences, unique among the alphoid DNAs, have been conservatively maintained throughout primate phylogeny and that they are among the most ancient of all primate DNAs.     

Ovine mitochondrial DNA: restriction enzyme analysis, mapping and sequencing data

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Rates of nuclear and cytoplasmic mitochondrial DNA sequence divergence in mammals

Differential rates of nucleotide substitution among different gene segments and between distinct evolutionary lineages is well documented among mitochondrial genes and is likely a consequence of locus-specific selective constraints that delimit mutational divergence over evolutionary time. We compared sequence variation of 18 homologous loci (15 coding genes and 3 parts of the control region) among 10 mammalian mitochondrial DNA genomes which allowed us to describe different mitochondrial evolutionary patterns and to produce an estimation of the relative order of gene divergence. The relative rates of divergence of mitochondrial DNA genes in the family Felidae were estimated by comparing their divergence from homologous counterpart genes included in nuclear mitochondrial DNA (Numt, pronounced "new might"), a genomic fossil that represents an ancient transfer of 7.9 kb of mitochondrial DNA to the nuclear genome of an ancestral species of the domestic cat (Felis catus). Phylogenetic analyses of mitochondrial (mtDNA) sequences with multiple outgroup species were conducted to date the ancestral node common to the Numt and the cytoplasmic (Cymt) mtDNA genes and to calibrate the rate of sequence divergence of mitochondrial genes relative to nuclear homologous counterparts. By setting the fastest substitution rate as strictly mutational, an empirical "selective retardation index" is computed to quantify the sum of all constraints, selective and otherwise, that limit sequence divergence of mitochondrial gene sequences over time.      

Mitochondrial genome of the colorless green alga Polytomella parva: two linear DNA molecules with homologous inverted repeat Termini

Most of the well-characterized mitochondrial genomes from diverse green algal lineages are circular mapping DNA molecules; however, Chlamydomonas reinhardtii has a linear 15.8 kb unit mitochondrial genome with 580 or 581 bp inverted repeat ends. In mitochondrial-enriched fractions prepared from Polytomella parva (=P. agilis), a colorless, naturally wall-less relative of C. reinhardtii, we have detected two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5 kb. Sequences spanning 97% and 86% of the 13.5- and 3.5-kb mtDNAs, respectively, reveal that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated nonsynonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA [LSU and SSU rRNA] genes, one tRNA gene, and six protein-coding genes). On the basis of DNA sequence data, we propose that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (=C. eugametos) as the outgroup, we conclude that the nonsynonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on an average about 3.3 times that of the C. reinhardtii counterparts.     

Identification and functional validation of a 5' upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non-coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5' upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at -15 kb. We detected several stretches of homology within the first 30 kb 5' tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5' upstream regulatory sequence found between -8 and -10 kb of the human tyrosinase locus (termed h5'URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at -9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue-specific enhancer in the h5'URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5'URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.     

Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.     

Interruption of an alpha-satellite array by a short member of the KpnI family of interspersed, highly repeated monkey DNA sequences.

We describe here the interruption of a cloned African green monkey alpha-satellite array by an 829-base-pair-long nonsatellite DNA segment. Hybridization experiments indicate that the sequences within the interruption are homologous to segments frequently found in the 6-kilobase-pair-long members of the KpnI family of long, interspersed repeats. These data confirm and extend earlier results suggesting that sequences common to the KpnI family can occur independently of one another and in segments of variable lengths. The 829-base-pair-long segment, which is termed KpnI-RET, contains a terminal stretch of adenosine residues preceded by two typical but overlapping polyadenylation sites. KpnI-RET is flanked by direct repeats of a 14-base-pair-long segment of alpha-satellite that occurs only once in the satellite consensus sequence. These structural features suggest that KpnI-RET was inserted into the satellite array as a movable element.     

Pankinetoplast DNA structure in a primitive bodonid flagellate, Cryptobia helicis.

The mitochondrial DNA (mtDNA) of a primitive kinetoplastid flagellate Cryptobia helicis is composed of 4.2 kb minicircles and 43 kb maxicircles. 85% and 6% of the minicircles are in the form of supercoiled (SC) and relaxed (OC) monomers, respectively. The remaining minicircles (9%) constitute catenated oligomers composed of both the SC and OC molecules. Minicircles contain bent helix and sequences homologous to the minicircle conserved sequence blocks. Maxicircles encode typical mitochondrial genes and are not catenated. The mtDNA, which we describe with the term 'pankinetoplast DNA', is spread throughout the mitochondrial lumen, where it is associated with multiple electron-lucent loci. There are approximately 8400 minicircles per pankinetoplast-mitochondrion, with the pan-kDNA representing approximately 36% of the total cellular DNA. Based on the similarity of the C.helicis minicircles to plasmids, we present a theory on the formation of the kDNA network.     

KpnI families of long, interspersed repetitive DNAs associated with the human β-globin gene cluster

KpnI families of long, interspersed repetitive DNAs are ubiquitous repetitive elements that occur in tens of thousands of copies in primate genomes. KpnI 1.2, 1.5 and two different KpnI 1.8-kb families were found within and flanking a 6.4-kb repeat beginning at 3 kb, 3' from the human β-globin gene. Thus, six different types of KpnI families have now been identified, and four of these are found next to each other in a specific 6.4-kb repeat. Clones of the distinct KpnI families were hybridized to clones of the 6.4-kb repeat and adjacent sequences encompassed within some 17.6 kb of DNA lying 3' to the β-globin gene cluster. The four KpnI families appear to make up the entire length of the 6.4-kb repeat. The linear order of the various cloned KpnI sequences in the repeat is 5'-pBK(1.8)26-pBK.(1.5)54-pBK(1.2)11-pBK(1.8)11-3'. KpnI 1.2-kb sequences were also detected downstream from the 6.4-kb repeat. As in the case of the KpnI 1.2 and 1.5-kb families, the two KpnI 1.8-kb sequence families described here each hybridized with about 15% of all plaques in two independently generated human genome libraries.