Members of the KpnI family of long interspersed repeated sequences join and interrupt alpha-satellite in the monkey genome.

Three different members of a family (KpnI-family) of interspersed repeated DNA sequences were found linked to alpha-satellite sequences in cloned segments of the African green monkey genome. In two of these segments the KpnI-family member is over 6 kbp in length and one of them is flanked by alpha-satellite on both sides indicating that it was inserted into a satellite array. Hybridization of subcloned portions of the family members to restriction endonuclease digests of monkey and human DNA and to a genomic library of African green monkey DNA indicate that 1) family members are interspersed in both the monkey and human genomes, 2) some family members may include sequences in addition to those in the three characterized here, 3) some family members may contain only parts of the sequences characterized here and 4) while the overall organization of the family is similar in the human and monkey genome the majority of the family members in each of the two genomes are distinctly identified by the variant position of certain restriction endonuclease sites. This last observation suggests that within each genome there is a tendency to maintain particular versions of the sequence. Observations 2) and 3) suggest that the KpnI family is complex and includes a variety of subfamilies.     

Interruption of a human nuclear sequence homologous to mitochondrial DNA by a member of the KpnI 1.8 kb family.

We reported that several DNA sequences homologous to mitochondrial DNA (mtDNA) are present in the human nuclear genome (Tsuzuki et al. (1983) Gene 25, 223-229). Detailed Southern blot analyses revealed that one of such sequences is interrupted by a repetitive sequence about 1.8 kb long, and that the insert is one member of the dispersed repeated DNA sequences of the KpnI 1.8 kb family. Nucleotide sequence analysis showed that the KpnI 1.8 kb DNA is flanked with imperfect 15-base pair (bp) direct repeats of mtDNA. This KpnI 1.8 kb DNA has an A-rich sequence at its 3'-end, and has a considerable homology with one of the published cDNA sequences homologous to one of the human KpnI families and also to one of the African green monkey KpnI families, KpnI-LS1. These structural features suggest that the KpnI 1.8 kb DNA is a movable element and is inserted within the mtDNA-like sequence by an RNA-mediated process.     

The sequence of the 3.3-kilobase repetitive element fromDipodomys ordii suggests a mechanism for its amplification and interspersion

Summary DNA from the kangaroo rat,Dipodomys ordii, contains a 3.3-kb, highly repeated sequence that is interspersed throughout the genome in small tandem clusters. One 3.3-kb unit has been cloned into pBR322 and the nucleotide sequence determined. The clone used was shown to be representative of the bulk of such sequences found in the genomic DNA. The sequence contains 10 homologous subunits each ca. 260 bp in length. Comparison of these to one another yielded a 258-bp consensus sequence containing a 35-bp terminal inverted repeat. Two unique stretches also occur. One of these contains a region that could serve as a promoter for RNA polymerase III; the other contains a sequence related to the ARS sequences of yeast. It is proposed that an ancestral sequence similar to the consensus sequence was amplified to 10 or more units, and that, subsequently, two other sequences were inserted. The properties of these insertions may have led to the dispersal of the sequence throughout the genome.     

Structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific RNA

We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.     

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.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B)

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).     

Defining the beginning and end of KpnI family segments.

Comparison of the sequences at the ends of several newly cloned and full length members of the monkey KpnI family with one another and with previously described monkey and human segments defines the nucleotide sequence at the two termini. No terminal repeats either direct or inverted are noted within full length family members which may or may not be immediately flanked by direct repeats. At the 3' terminus, several family members have polyadenylation signals followed by a d(A)-rich stretch. The genomic frequency of segments within the full length element increases markedly from the 5' to the 3' terminus, consistent with the cloning of various truncated family members. One such truncated version joined to a low copy number DNA segment is inserted in monkey alpha-satellite where the combination appears to have been amplified in conjunction with the satellite itself.     

Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1 at the 5' and 3' sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.     

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.     

The normal developmental regulation of a cloned sgs3 ''glue'' gene chromosomally integrated in Drosophila melanogaster by P element transformation.

A 7-kb genomic segment containing the coding sequence for the Drosophila melanogaster Formosa variant of salivary gland secretion protein 3 (sgs3) has been inserted into the snw y, bw, st strain of D. melanogaster using the P transformation vector p.6.1. The inserted sequence codes for a shorter RNA which can be distinguished from that of the host gene. The amount of RNA, and its stage- and tissue-specific synthesis is identical to that of the host gene, which suggests that all the cis-acting regulatory DNA sequences for this gene are contained within this 7-kb segment.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase

KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.     

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.     

Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

The gene coding for alpha-amylase from Bacillus amyloliquefaciens was isolated by direct shotgun cloning using B. subtilis as a host. The genome of B. amyloliquefaciens was partially digested with the restriction endonuclease MboI and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. One of the transformants producing high amounts of alpha-amylase was characterized further. The alpha-amylase gene was shown to be present in a 2.3-kb insert. The alpha-amylase production of the transformed B. subtilis could be prevented by inserting lambda DNA fragments into unique sites of EcoRI, HindIII and KpnI in the insert. Foreign DNA inserted into a unique ClaI site failed to affect the alpha-amylase production. The amount of alpha-amylase activity produced by this transformed B. subtilis was about 2500-fold higher than that for the wild-type B. subtilis Marburg strain, and about 5 times higher than the activity produced by the donor B. amyloliquefaciens strain. Virtually all of the alpha-amylase was secreted into the culture medium. The secreted alpha-amylase was shown to be indistinguishable from that of B. amyloliquefaciens as based on immunological and biochemical criteria.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.