Analysis of the primary structure of DNA sequences flanking MDG1

MDG is a very important component of the Drosophila genome. MDG have many sites of localisation in chromosomes and can change their localisation. Perhaps the process of MDG integration has some specificity. To study this problem we sequenced the flanking region of MDG1 DNA. The analysis of this sequences reveals the following features. 1. The 5'-flanking sequences contain 7 TATA-boxex, 5 of which form a cluster. 2. The 3'-flanking sequences contain TTTAAA block which is similar to TATA-box for alpha- and gamma-casein genes of mammals. 3. The flanking region are rich in repeated sequences, the longest of which TCCTCCT (R) and TTCTTC (R2) are on the 5'-flank and on the 3'-flank respectively, so that the whole structure is: 5'-R1NNR1-MDG1-R2NNR2-3', where N is some nucleotide. 5'-flanking sequences are AT-rich, while the 3'-flank contains 10 consecutive thymidines 4 nucleotides apart from MDG1. The MDG1 and MDG "17.6" share several common repeats in the flanking sequences, the longest of which TACTTACAT is 63 bases upstream MDG1 and 11 bases upstream MDG "17.6". This sequence differs strongly from the consensus enhancer sequence.     

Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1)

Seven out of twenty 30–50 kb genome fragments with an MDG1 copia-like element cloned in cosmids were found to carry homologous sequences which belong to a new family of non-mobile heterochromatic moderate repeats (the HMR family). These repeats along with the MDG1 copies inserted in them are under-replicated in polytene chromosomes. Such repeats may also be located in the intercalary heterochromatin site 12E of the X chromosome. Chromosomal heterochromatic regions are enriched with one of the two main genomic variants of MDG1, MDG1^het, identifiable by EcoRI restriction. From Southern DNA blot analysis the number of MDG1^het copies and their sites within the heterochromatin are invariant in all the stocks examined, while there is not a single MDG1 site along the polytene chromosomes shared by all the stocks in question.     

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.     

Maintenance of the copy number of retrotransposon MDG3 in the Drosophila melanogaster genome]

The genomes of laboratory stocks and natural population of Drosophila melanogaster contain 8-12 copies of retrotransposon MDG3 detected by in situ hybridization. Construction of genotypes with decreased MDG3 copy number using X-chromosome and chromosome 3 free of MDG3 copies results in appearance of hybrid genomes carrying up to 7-10 copies, instead of 2-4 copies expected. New MDG3 copies are detected in different genome regions, including the 42B hot spot of their location. The chromosomes, where new clusters of MDG3 were observed, carry conserved "parental pattern" of MDG1 arrangement. The data obtained suggest the existence of genomic mechanism for maintenance of retrotransposon copy number on a definite level.     

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM.     

Mobile genetic element MDG4 (gypsy) in Drosophila melanogaster. Features of structure and regulation of transposition]

Distribution of two structural functional variants of the MDG4 (gypsy) mobile genetic element was examined in 44 strains of Drosophila melanogaster. The results obtained suggest that less transpositionally active MDG4 variant is more ancient component of the Drosophila genome. Using Southern blotting, five strains characterized by increased copy number of MDG4 with significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize MDG4 independently from the cellular flamenco gene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam- mutant allele causing active transpositions of the MDG4. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained.     

RNA-DNA hybrids containing damaged DNA are substrates for RNase H

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.     

Cleavage of DNA.RNA hybrids by type II restriction enzymes.

The action of a number of restriction enzymes on DNA.RNA hybrids has been examined using hybrids synthesised with RNAs of cucumber mosaic virus as templates. The enzymes EcoRI, HindII, SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strand of the hybrids (and possible also the RNA strand) into specific fragments. For four of these enzymes, HhaI, AluI, TaqI and HaeIII, comparison of the restriction fragments produced with the known sequences of the viral RNAs confirmed that they were recognising and cleaving the DNA strand of the hybrids at their correct recognition sequences. It is likely that the ability to utilise DNA.RNA hybrids as substrates is a general property of Type II restriction enzymes.     

Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon

The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.     

The Distribution in Different Strains and Characteristic Features of Two Subfamilies of Drosophila melanogaster Retrotransposon MDG4 (gypsy)

The distribution of two variants of MDG4 (gypsy) was analyzed in severalDrosophila melanogasterstrains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutantflamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovo^D test.     

Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3'processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3'-hydroxyl group of 2'-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2'-deoxyadenosine containing a 3'-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.