The elite athlete and exercise in pregnancy

To construct an effective recombinant Marek's disease virus type 1 (MDV1), we localized a stable insertion site for expression of the Escherichia coli lacZ gene near or within the short inverted repeats of MDV1 strain K554 DNA. A stable recombinant MDV1 was obtained by deleting the junction region between the short unique sequence (Us) and the internal short inverted repeat (IRs). The recombinant MDV1 replicated in cultured cells as well as the parental viral DNA. Antibodies against both MDV1 antigen and beta-galactosidase encoded by the lacZ gene were detected in the sera of chickens immunized with the virus, and persisted for at least 16 weeks. Moreover, the recombinant virus conferred protection upon chickens against a challenge with virulent MDV1. These results demonstrated that the Us-IRs junction region is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry. Analysis of the Us-IRs junction region which was deleted from the parental MDV1 indicated that there is a tandem direct repeat of a 220-bp exists within the short internal and terminal inverted repeats of avirulent MDV1 K554 strain DNA. The 220-bp sequence was well conserved among DNAs from various strains. The number of the repeat units may differ between the IRs and TRs or among various MDV1 strain DNAs.     

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts

Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.     

Stability of an inverted repeat in a human fibrosarcoma cell

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

IS481 and IS1002 of Bordetella pertussis create a 6-base-pair duplication upon insertion at a consensus target site

The insertion sequence IS481 and its isoform IS1002 have been observed to transpose into the bvgAS locus of Bordetella pertussis, for which the DNA sequence has previously been determined. Upon insertion of IS481 at three different sites and IS1002 at one site, a 6-bp sequence originally present was found at the junction of bvg and insertion sequence DNA. This indicates that, contrary to prior reports, IS481 and IS1002 do create a duplication upon insertion. In this light, examination of these and other examples of IS481 and IS1002 reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS481 or IS1002, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.     

Evolutionary dynamics of tandem repeats in the mitochondrial DNA control region of the minnow Cyprinella spiloptera

Length variation due to tandem repeats is now recognized as a common feature of animal mitochondrial DNA; however, the evolutionary dynamics of repeated sequences are not well understood. Using phylogenetic analysis, predictions of three models of repeat evolution were tested for arrays of 260-bp repeats in the cyprinid fish Cyprinella spiloptera. Variation at different nucleotide positions in individual repeats supported different models of repeat evolution. One set of characters included several nucleotide variants found in all copies from a limited number of individuals, while the other set included an 8-bp deletion found in a limited number of copies in all individuals. The deletion and an associated nucleotide change appear to be the result of a deterministic, rather than stochastic, mutation process. Parallel origins of repeat arrays in different mitochondrial lineages, possibly coupled with a homogenization mechanism, best explain the distribution of nucleotide variation.     

Circularization and cleavage of guinea pig cytomegalovirus genomes

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.     

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions

Using polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNALeu(UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent). Two-thirds of the rearrangements terminated at short (3-9-bp) direct repeats. Three of the different rearrangements were detected in more than one animal; the most common rearrangement was found in nine different template preparations. Two loci (hot spots) were found to be particularly susceptible to rearrangement, and both were located at sequences that exhibited highly conserved potential for secondary structure formation. The displacement loop region of 10 samples exhibited the presence of multiple tandem duplications ranging between 324 and 449 bp in length. One of these consisted of heterologous, but overlapping, repeating units. Identical PCR protocols were carried out in control experiments using a cloned fragment of mtDNA that encompassed the most common hot spot sequence. The results showed that this fragment did not artifactually generate a rearrangement junction under our PCR conditions and suggested that this sequence does not promote rearrangement mutations in bacteria during the cloning process.     

Nucleotide sequence and genomic organization of repeating units of the AT-rich family or repeats in the diploid wheat Triticum monococcum L

In diploid wheat Triticum monococcum L., a novel family of AT-rich repeated DNA sequences arranged in clusters of tandem arrays of 560-bp fragments is described. The nucleotide sequence of a 556-bp repeat unit was determined. The basic structural unit of this repeated DNA family contains subrepeats with the main motif AAACATTGTTAC. This type of repetitive DNA sequence could evolve via tandem amplification of short subrepeats with some subsequent divergence, and/or via recombination with unique sequences. Structural organization of homologous sequences, revealed by blot hybridization in other representatives of the Triticeae tribe, suggest that this family of repeated DNA is genome-specific.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C alpha 1 and C alpha 2 genes

We have isolated sequences downstream of human Ig C alpha 1 and C alpha 2 genes and have identified two enhancers in these regions. One enhancer is located approximately 9 kb downstream of C alpha 1, and the second enhancer is located approximately 11 kb downstream of C alpha 2. These approximately 1.6-kb enhancers are virtually identical to each other except for varying numbers of a approximately 53-bp motif. The C alpha 2-associated enhancer contains four copies of this motif in tandem, whereas the C alpha 1-associated enhancer has only a single copy. Within the human enhancers is a 177-bp segment that is homologous to a 191-bp segment of one of four enhancers from the 3' regulatory region of murine (and rat) DNA, namely 3'IgH-E(hs1,2). Like the murine and rat enhancers, both human enhancers are flanked by inverted repeats; furthermore, the human enhancers generally appear to be inverted with respect to each other. The evolutionarily conserved region of homology has substantial core enhancer activity. Contained within this region are the single octamer and one copy of the approximately 53-bp motif, both of which contribute to the activity of the full-length enhancer. A comparison of the DNA sequences and the results of transient transfection assays imply that the human C alpha-associated enhancers may be regulated (in part) differently than the murine enhancer 3'IgH-E(hs1,2).     

The structures of integration sites in transgenic rice

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.     

Molecular structure of a wheat chromosome end healed after gametocidal gene-induced breakage

In the progeny of the monosomic addition line of common wheat, Triticum aestivum, carrying the gametocidal chromosome of Aegilops cylindrica, deletion chromosomes carrying the break point within the nucleolar organizing region of chromosome 1B appeared. Attempts were made to amplify the break points by PCR using primers of telomere and rDNA (rRNA gene). In one deletion line, specific amplification of DNA fragments including the 18S rRNA gene, telomere repeats, and their junction occurred. At the junction of telomere and rRNA gene there was a 31-bp inverted duplication of the rRNA gene. Telomere sequences were initiated from the sequence TAG in the duplication. Between the duplications a small sequence was also inserted. This novel DNA sequence at the break point indicates that the breakage-fusion-bridge cycle(s) took place after the first chromatin breakage by the gametocidal gene.     

The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction

Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.