A recombination hotspot in the LTR of a mouse retrotransposon identified in an in vitro system

The recombinational frequency between two long terminal repeat elements (LTR-IS) of a mouse retrotransposon was about 13 times higher, compared with that of two control DNA sequences in extracts from mouse testes, but not in extracts from ascites cells. Deletion of a 37 bp region from the LTR-IS element strongly suppresses its recombinational activity. This 37 bp region encompasses an area of potentially single-stranded DNA and interacts with at least two nuclear proteins. One of them binds sequence-specifically to single-stranded DNA and is present in both types of extracts. Another protein(s) binds to dsDNA at the motif TGGAAATCCCC and is absent in extracts from testes. Our results suggest that a cis-acting DNA sequence within the 504 bp LTR-IS element is responsible for its high recombinational activity in vitro, and they further support the previous suggestion that the LTR-IS elements are meiotic recombinational hotspots in vivo.     

Evidence for mobility of a new family of mouse middle repetitive DNA elements (LTR-IS)

Locus variation and sequence conservation of mouse LTR-IS elements, a new family of middle repetitive DNA sequences was studied. It is shown that LTR-IS sequences are present in all the inbred strains and subspecies of M. musculus tested and in M. cooki and M. caroli. Their arrangement in mouse genomes is polymorphic. Southern blot analysis and DNA sequencing revealed the existence of homologous DNA sequences with and without LTR-IS element insertion. LTR-IS sequences therefore appear to have arisen in early mouse ancestors and have, at least at some point, been mobile.     

Functional RNA polymerase II promoters in solitary retroviral long terminal repeats (LTR-IS elements).

LTR-IS elements are middle repetitive sequences in the mouse genome with structural features of solitary retroviral LTRs. In order to get some insight in the possible functional role of these sequences the promotor activity of two LTR-IS representatives differing by 105 bp in their U3 region was investigated. Gene fusions between LTR-IS sequences and the bacterial gene coding for chloramphenicol acetyl transferase (CAT) were transfected into mouse 3T6 cells and the expression of CAT was measured. It is shown that the LTR-IS sequences represent weak RNA polymerase II promoters which require enhancement by cis-or trans-activating factors.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Amplification of a chorion gene cluster in Drosophila is subject to multiple cis-regulatory elements and to long-range position effects

We have used P-element transformation to study cis-acting elements involved in the control of amplification of the third chromosome chorion gene cluster (66D12-15) in Drosophila melanogaster. To reduce position effects large fragments (5.7 to 12 kb; kb = 10(3) bases) of chorion DNA and the 7.2 kb ry+ fragment were used to "buffer" these putative elements from sequences at the insertion site. Nevertheless, even the longest constructs were profoundly affected by the insertion sites and showed amplification levels ranging from undetectable to higher than in the endogenous locus. Any amplification was tissue and temporally correct and extended into the neighboring ry+ sequences. Analysis of amplification levels at various points along two constructs bearing the same 10 kb chorion insert in opposite orientations showed maximal levels occurring at one end of the chorion fragment, irrespective of whether that end was buffered at the middle of the transposon or exposed close to the insertion site. The maximally amplifying region encompasses the amplification control element (ACE), which has been shown to be necessary for amplification, in agreement with its putative role as a replication origin. We have additionally identified amplification-enhancing elements present elsewhere in the 10 kb chorion fragment, which are needed for attainment of high copy number. These elements, distinct from the ACE, have been only coarsely localized within two 2.25 to 2.3 kb regions. Some interesting sequence similarities between these two regions and the ACE element are pointed out.     

Transforming Sloan-Kettering viruses generated from the cloned v-ski oncogene by in vitro and in vivo recombinations.

The Sloan-Kettering viruses (SKVs) are replication-defective retroviruses that transform avian cells in vitro. Each of the three SKV isolates is a mixture of viruses with genomes ranging in size from 4.1 to 8.9 kilobases (kb) with a predominant genome of 5.7 kb. Using a cDNA representing a sequence, v-ski, that is SKV specific and held in common by the multiple SKV genomes, we generated a restriction map of the 5.7-kb SKV genome and molecularly cloned a ski-containing fragment from SKV proviral DNA. Southern hybridization and sequence analysis showed that the cloned DNA fragment consisted of the 1.3-kb ski sequence embedded in the p19gag sequence and followed by the remaining 5' half of the gag gene and small portions of both the pol and env genes. A large deletion encompassing the 3' half of gag and the 5' 80% of pol was mapped to a position about 1 kb downstream from the 3' ski-gag junction. To determine whether the cloned ski sequence had transforming activity, the ski-containing fragment and a cloned Rous-associated virus 1 (RAV-1) genome were used to construct an analog of the 5.7-kb SKV genome, RAV-SKV. Cotransfection of chicken embryo cells with RAV-SKV and RAV-1 yielded foci of transformed cells whose morphology was identical to that induced by the natural SKVs. The transformed transfected cells produced transforming virus with a 5.7-kb ski-containing genome and synthesized a gag-containing polyprotein of 110 kilodaltons (kDa). Several nonproducer clones of RAV-SKV-transformed cells were analyzed, and most were found to synthesize a 5.7-kb SKV RNA and a 110-kDa polyprotein. One clone was found to contain an 8.9-kb SKV RNA, and this clone synthesized a 125-kDa polyprotein. Since both the 5.7- and 8.9-kb genomes and the 110- and 125-kDa polyproteins had been identified in studies on the natural SKVs, the present results not only demonstrate the transforming activity of these individual SKVs but also suggest mechanisms for their generation.     

Cloning and nucleotide sequence of a linear DNA plasmid fromXanthophyllomyces dendrorhous (Phaffia rhodozyma)

Extrachromosomal elements were found in a strain ofX. dendrorhous, and were characterized as linear DNA forming two well defined groups, pPh1 with 3 high-copy-number molecules, pPh11 (6.9 kb), pPh12 (5.7), pPh13 (4.7), and pPh2 with 2 low-copy-number molecules, pPh21 (3.6 kb), pPh22 (3.0). A 4077 bp fragment from pPh13 was cloned in pUC18 (pDK1) and sequenced (accession no. AJ 278 424). Seven putative ORF and some possible regulator sequences were defined.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region,the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.