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The Enterococcus faecalis pathogenicity island (PAI) encodes known virulence traits and >100 additional genes with unknown roles in enterococcal biology. Phage-related integration and excision genes, and direct repeats flanking the island, suggest it moves as an integrative conjugative element (ICE). However, transfer was observed not to require these genes. Transfer only occurred from donors possessing a pheromone responsive-type of conjugative plasmid, and was invariably accompanied by transfer of flanking donor chromosome sequences. Deletion of plasmid transfer functions, including the cis -acting origin of transfer ( oriT ), abolished movement. In addition to demonstrating PAI movement by a mechanism involving plasmid integration, we observed transfer of a selectable marker placed virtually anywhere on the chromosome. Transfer of this selectable marker was observed to be accompanied by chromosome-chromosome transfer of vancomycin resistance, MLST markers, and capsule genes as well. Plasmid mobilization therefore appears to be a major mechanism for horizontal gene transfer in the evolution of antibiotic resistant E. faecalis strains capable of causing human infection.

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis . We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10–18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination.

Interspersed repeat composition and distribution in mammals have been best characterized in the human and mouse genomes. The bovine genome contains typical eutherian mammal repeats, but also has a significant number of long interspersed nuclear element RTE (BovB) elements proposed to have been horizontally transferred from squamata. Our analysis of the BovB repeats has indicated that only a few of them are currently likely to retrotranspose in cattle. However, bovine L1 repeats (L1 BT) have many likely active copies. Comparison of substitution rates for BovB and L1 BT indicates that L1 BT is a younger repeat family than BovB. In contrast to mouse and human, L1 occurrence is not negatively correlated with G+C content. However, BovB, Bov A2, ART2A, and Bov-tA are negatively correlated with G+C, although Bov-tAs correlation is weaker. Also, by performing genome wide correlation analysis of interspersed and simple sequence repeats, we have identified genome territories by repeat content that appear to define ancestral vs. ruminant-specific genomic regions. These ancestral regions, enriched with L2 and MIR repeats, are largely conserved between bovine and human.

Direct repeats of Arabidopsis telomeric sequence were constructed to test telomere-mediated chromosomal truncation in maize. Two constructs with 2.6 kb of telomeric sequence were used to transform maize immature embryos by Agrobacterium -mediated transformation. One hundred seventy-six transgenic lines were recovered in which 231 transgene loci were revealed by a FISH analysis. To analyze chromosomal truncations that result in transgenes located near chromosomal termini, Southern hybridization analyses were performed. A pattern of smear in truncated lines was seen as compared with discrete bands for internal integrations, because telomeres in different cells are elongated differently by telomerase. When multiple restriction enzymes were used to map the transgene positions, the size of the smears shifted in accordance with the locations of restriction sites on the construct. This result demonstrated that the transgene was present at the end of the chromosome immediately before the integrated telomere sequence. Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends. These results demonstrate that telomere-mediated chromosomal truncation operates in plant species. This technology will be useful for chromosomal engineering in maize as well as other plant species.

Botulinum neurotoxins (BoNTXs) produced by Clostridium botulinum are among the most poisonous substances known. Of the seven types of BoNTXs, genes for type C1 and D toxins (BoNTX/C1 and D) are carried by bacteriophages. The gene for exoenzyme C3 also resides on these phages. Here, we present the complete genome sequence of c-st, a representative of BoNTX/C1-converting phages. The genome is a linear double-stranded DNA of 185,682 bp with 404-bp terminal direct repeats, the largest known temperate phage genome. We identified 198 potential protein-coding regions, including the genes for production of BoNTX/C1 and exoenzyme C3. Very exceptionally, as a viable bacteriophage, a number of insertion sequences were found on the c-st genome. By analyzing the molecular structure of the c-st genome in lysogens, we also found that it exists as a circular plasmid prophage. These features account for the unstable lysogeny of BoNTX phages, which has historically been called “pseudolysogeny.” The PCR scanning analysis of other BoNTX/C1 and D phages based on the c-st sequence further revealed that BoNTX phages comprise a divergent phage family, probably generated by exchanging genomic segments among BoNTX phages and their relatives.

Myotonic dystrophy (DM) type 1 is associated with an expansion of (>50) CTG repeats within the 3′ untranslated region (UTR) of the dystrophin myotonin protein kinase gene ( dmpk ). In the corresponding mRNA transcript, the CUG repeats form an extended stem-loop structure. The double-stranded RNA of the stem sequesters RNA binding proteins away from their normal cellular targets resulting in aberrant transcription, alternative splicing patterns, or both, thereby leading to DM. To better understand the structural basis of DM type 1, we determined to 1.58-Å resolution the x-ray crystal structure of an 18-bp RNA containing six CUG repeats. The CUG repeats form antiparallel double-stranded helices that stack end-on-end in the crystal to form infinite, pseudocontinuous helices similar to the long CUG stem loops formed by the expanded CUG repeats in DM type 1. The CUG helix is very similar in structure to A-form RNA with the exception of the unique U-U mismatches. This structure provides a high-resolution view of a toxic, trinucleotide repeat RNA.

Steroid receptors bind as dimers to a degenerate set of response elements containing inverted repeats of a hexameric half-site separated by 3 bp of spacer (IR3). Naturally occurring selective androgen response elements have recently been identified that resemble direct repeats of the hexameric half-site (ADR3). The 3D crystal structure of the androgen receptor (AR) DNA-binding domain bound to a selective ADR3 reveals an unexpected head-to-head arrangement of the two protomers rather than the expected head-to-tail arrangement seen in nuclear receptors bound to response elements of similar geometry. Compared with the glucocorticoid receptor, the DNA-binding domain dimer interface of the AR has additional interactions that stabilize the AR dimer and increase the affinity for nonconsensus response elements. This increased interfacial stability compared with the other steroid receptors may account for the selective binding of AR to ADR3 response elements.

Many overlapping surveillance and repair mechanisms operate in eukaryotic cells to ensure the stability of the genome. We have screened to isolate yeast mutants exhibiting increased levels of recombination between repeated sequences. Here we characterize one of these mutants, elg1 . Strains lacking Elg1p exhibit elevated levels of recombination between homologous and nonhomologous chromosomes, as well as between sister chromatids and direct repeats. These strains also exhibit increased levels of chromosome loss. The Elg1 protein shares sequence homology with the large subunit of the clamp loader replication factor C (RFC) and with the product of two additional genes involved in checkpoint functions and genome maintenance: RAD24 and CTF18 . Elg1p forms a complex with the Rfc2–5 subunits of RFC that is distinct from the previously described RFC-like complexes containing Rad24 and Ctf18. Genetic data indicate that the Elg1, Ctf18, and Rad24 RFC-like complexes work in three separate pathways important for maintaining the integrity of the genome and for coping with various genomic stresses.

TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix–loop–helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c- fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c- fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.

A cell culture system has been used to determine the relative rates of insertions and deletions of integral numbers of dinucleotide repeats in a microsatellite sequence. A plasmid was constructed that contained 17 repeats of poly(dC-dA)⋅poly(dG-dT) near the 5′ end of a bacterial neomycin-resistance ( neo) gene, such that the neo gene was translated in the (+1) reading frame. The plasmid was introduced into mismatch-repair-proficient and mismatch-repair-deficient mammalian cell lines. Rates of mutation to resistance to the neomycin analogue G418 were measured, and the nature of the mutations was determined. The mutations were all gains or losses of integral numbers of repeats, and mutations involving a single repeat greatly predominated over those involving multiple repeats. The data obtained from these studies were compared with results previously obtained with cells transfected with a similar plasmid in which the sequence of the oligonucleotide insert placed the neo gene in the (−1) reading frame. This experimental design made it possible to make direct comparisons between insertions and deletions of a single repeat unit. A significant excess of insertions over deletions was found in both repair-proficient and repair-deficient cell lines, although the few mutations involving more than two repeats were deletions.

The pir gene of plasmid R6K encodes the protein, π, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of π protein: a 35.0-kDa form (π 35.0 ) and a shortened 30.5-kDa form (π 30.5 ). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only π 35.0 can bind to a site in the (A⋅T)-rich segment of its γ ori and activate the γ ori in vivo and in vitro . However, unlike π 35.0 , π 30.5 can inhibit in vivo and in vitro replication (activated by π 35.0 ). We propose that the two forms of π might have distinct functions in replication. We show that although both forms of π produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable π 35.0 dimers, whereas another, apparently weaker interface holds together dimers of π 30.5 . We speculate that the leucine zipper-like motif, absent in π 30.5 , controls very specific functions of π protein.

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.

Using computer programs developed for this purpose, we searched for various repeated sequences including inverted, direct tandem, and homopurine–homopyrimidine mirror repeats in various prokaryotes, eukaryotes, and an archaebacterium. Comparison of observed frequencies with expectations revealed that in bacterial genomes and organelles the frequency of different repeats is either random or enriched for inverted and/or direct tandem repeats. By contrast, in all eukaryotic genomes studied, we observed an overrepresentation of all repeats, especially homopurine–homopyrimidine mirror repeats. Analysis of the genomic distribution of all abundant repeats showed that they are virtually excluded from coding sequences. Unexpectedly, the frequencies of abundant repeats normalized for their expectations were almost perfect exponential functions of their size, and for a given repeat this function was indistinguishable between different genomes.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

A global analysis of the 230-kilobase-pair (kbp) human cytomegalovirus genome revealed three regions that were very rich in repeated sequences. The region with the highest content of inverted and direct repeats lies between 92,100 and 93,500 bp, upstream of the gene encoding the single-stranded DNA binding protein. Cloned restriction fragments containing this region were able to replicate when trans-acting factors were provided by virus infection in a transient replication assay. With this assay, the region between 92,210 and 93,715 bp on the viral genome was defined as the minimal replication origin, oriLyt. The sequence composition and repeats within oriLyt were used to divide the region into two domains that may be important in origin function. Sequences flanking either the left or right side of the minimal oriLyt contributed to efficient replication; however, these sequences were not essential for origin function. Thus, the region of the viral genome with the most striking concentration of direct and inverted repeats corresponds to the oriLyt of human cytomegalovirus.

We report the identification and partial characterization of the promoters for two chitinase genes from Streptomyces plicatus. Chitinases are a family of enzymes made by Streptomyces and other soil microbes to digest chitin, an abundant source of carbon and nitrogen in the soil. The promoter regions of two chitinases were defined by using transcriptional fusions to the xylE reporter gene. Transcription was shown to be glucose-sensitive and chitin-dependent. Each promoter contains a putative RNA polymerase binding site with a recognition sequence very similar to that observed in many eubacterial vegetatively expressed genes. In both promoters, a pair of 12-base-pair direct repeat sequences overlap the putative RNA polymerase binding sites. Further analysis of one of the promoters revealed that a single-base change within the direct repeat sequences resulted in glucose-resistant, chitin-independent expression in vivo. In addition, the promoter region that includes the direct repeat sequences was shown to interact with a sequence-specific DNA binding factor in vitro. Similar direct repeat sequences are present in other chitinase genes recently characterized, and we suggest that these repeats may be involved in repression and induction for this entire class of catabolite-controlled genes.

The involucrin genes of the prosimian primates and of the anthropoid primates possess nonhomologous segments of repeats located at two different sites, P and M, within the coding region. The involucrin gene of the tarsioids alone contains repeats at both sites, for it derived repeats at site P from a common ancestor of tarsioids and prosimians and a repeat at site M from a later common ancestor of tarsioids and anthropoids. After their divergence from the tarsioids, the anthropoids added many more repeats to site M and excised the older segment of repeats from site P; in contrast, the tarsioids stopped adding repeats at site M, retained the earlier segment of repeats at site P, and enlarged it. In the revision of their involucrin genes, the two lineages followed alternative routes. The mechanisms by which the revisions took place have been subject to abrupt onset or termination.

We determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus (SNV). A method was developed to clone integrated proviruses of retroviral shuttle vectors by exploiting the tight binding of the lac operator to the lac repressor protein. The vectors contained the lacZ alpha gene as a reporter of mutations. Thirty-seven of the 16,867 proviruses recovered contained five classes of mutations, including substitutions and frameshifts. Runs of 9 and 10 identical base pairs and a direct repeat of 110 base pairs were mutational hotspots. In addition, two copies of a provirus contained 15 G-to-A substitutions. Such proviruses, which we name hypermutants, may arise through the action of an error-prone polymerase and could significantly contribute to the genetic variation in retroviral populations.

Cloning of the overlapping DNA fragments together with Southern hybridization experiments showed the organization of the human C epsilon and C alpha gene cluster as 5'-C epsilon 2-14 kilobases-C alpha 1----C epsilon 1-13 kilobases-C alpha 2-3'. Comparison of the nucleotide sequences of the C epsilon 1 and C epsilon 2 genes revealed that four deletions have taken place in the C epsilon 2 gene and its flanking regions. The three deleted regions in the 5' side of the C epsilon 2 gene are partially filled with shorter inserted sequences. One of them has removed the CH1 and CH2 exons and a portion of the epsilon switch (S epsilon) region. The S epsilon region and the CH4 exon still retain the functional structures, whereas the CH3 exon has been inactivated by deleting its 5' intervening sequence necessary for splicing. The tetranucleotide T-G-G-G (or T-G-G-C), which is usually found in close proximity of the class-switch recombination sites in mouse myelomas, is located 5' to the three deletion sites. The results imply that the mechanism responsible for the heavy chain class-switch recombination might be relevant to the evolutionary mechanism of creation of the truncated C epsilon 2 gene. The other deletion in the 3' flanking region of the C epsilon 2 gene may be due to slipped mispairing of the short direct repeat (C-C-C-C-C) at both ends.

This paper identifies all statistically significant dyad symmetry combinations in the Epstein-Barr virus genome. The distribution of long dyad symmetry pairings emphasizes two regions, the 5' third of the 3.1-kilobase-pair (kbp) repeat and the oriP region, the latter essential for Epstein-Barr virus replication during latency. A 600-base-pair (bp) stretch in the 3.1-kbp repeat can establish an extended hairpin loop of stem length in excess of 208 bp of predominantly G + C stacking. Moreover, the 3.1-kbp repeat has the potential to form a wide variety of secondary structures based on juxtapositions of sizable palindromes, close dyad symmetry pairings, and direct repeats. The 3.1-kbp repeat presents several features that portend it as an important control region. The oriP region contains an abundance of statistically significant dyad symmetry combinations that strongly correlate with the "21 X 30 bp" tandem repeat units and four truncated copies of this repeat unit 1 kbp downstream. Each of the units centers on the same approximately 30-bp palindrome. Contrasts in the content and the secondary structure formations associated with the 3.1-kbp repeat units versus those of the oriP region are discussed in relation to viral or cellular function.

Experiments presented here show that each of the 1.5-kilobase inverted repeats of the kanamycin-resistance transposon Tn5 is transposable; we designate them IS50-L (left) and IS50-R (right). By DNA sequence analyses, IS50 is 1533 base pairs (bp) long and generates 9-bp direct repeats of target sequences. The ends of IS50 comprise a hyphenated 8-of-9-bp inverted repeat and are not used with equal efficiency; the outside ends are more active than the inside ends, suggesting that a strong transposase recognition site at the outside ends, suggesting that a strong transposase recognition site at the outside end extends beyond the 8 bp common to both ends.

A directed mutagenesis method has been developed for the analysis of mutagen specificity. The method is based on the construction of a plasmid damaged by the mutagen at a specific segment within a given marker gene, followed by screening for mutant plasmids and nucleotide sequence analysis of the damaged segment. By using this method plasmid pXf3 has been specifically damaged by UV radiation at the BamHI-Sph I segment in the tetracycline resistance (tet) gene(s) and used to transform SOS-induced Escherichia coli. Fourteen ampicillin-resistant, tetracycline-sensitive mutants of pXf3 were isolated and subjected to sequence analysis. The data revealed the induction of transitions, a transversion, a frameshift mutation, and deletions. The single base changes all were located within runs of pyrimidines, and the deletions mapped between direct repeats of polypyrimidine tracts. In addition, mutant plasmids were found with no mutation within the damaged segment. In these cases it is likely that "untargeted mutagenesis" in other portions of the tet gene(s) is responsible for the mutant phenotype. The method can be applied to any mutagen that reacts with DNA, and it also can be used for genetic analysis as a means to mutate specific segments of DNA.

The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae. The 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA. The 5' end is 245 bases into the left delta sequence measured from the left side of the Ty1 element. The delta sequence is a direct repeat of about 340 base pairs present at each end of the Ty1 element. The Ty1 transcription includes 93-97 bases of the left delta sequence and continues through the entire internal portion of the element and through about 295 bases of the right delta sequence before reaching the 3' end located 38-46 bases from the right side of the right delta sequence. Because the delta sequences present at each end of a single Ty1 element have identical or very similar DNA sequences, these end points for Ty1 RNA raise several questions about the expression of Ty1 elements. First, what are the initiation and termination signals, because the Ty1 transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Ty1 element opposite to that of genes that are overexpressed after the insertion of a Ty1 element? Third, because the Ty1 RNA itself has direct repeats of about 45 bases, a structure analogous to retrovirus RNAs, is the Ty1 RNA an intermediate in the transposition of Ty1?

We discovered the presence of an Alu and an Xba repetitive DNA element within introns 4 and 7, respectively, of the human alpha-fetoprotein (AFP) gene; these elements are absent from the same gene in the gorilla. The Alu element is flanked by 12-base-pair direct repeats, AGGATGTTGTGG ... (Alu) ... AGGATGTTGTGG, which presumably arose by way of duplication of the intronic target site AGGATGTTGTGG at the time of the Alu insertion. In the gorilla, only a single copy of the unoccupied target site is present, which is identical to the terminal repeat flanking the human Alu element. There are two copies of an Xba repeat in the human AFP gene, apparently the only two in the genome. Xba1 and Xba2, located within introns 8 and 7, respectively, differ from each other at 3 of 303 positions. Xba1 is referred to as the old (ancestral) repeat because it lacks direct repeats. The new (derived) Xba2 is flanked by direct repeats, TTTCTTTTT ... (Xba) ... TTTCTTCTT, and is thought to have arisen as a result of transposition of Xba1. The ancestral Xba1 and a single copy of the Xba2 target site are present at orthologous positions in the gorilla, but the new Xba2 is absent. We conclude that the Alu and Xba DNA repeats emerged in the human genome at a time postdating the human-gorilla divergence and became established as genetic novelties in the human lineage. We submit that the chronology of divergence of primate lines of evolution can be correlated with the timing of insertion of new DNA repeats into the genomes of those primates.

The nucleotide sequence of the long terminal repeat (LTR) of bovine leukemia virus, a unique oncogenic retrovirus of cattle, was determined. The LTR consisted of 530 base pairs (bp) with an inverted repeat of 6 bp at its 5' and 3' ends, flanked by a direct repeat of 6 bp of host cell origin. A tRNAPro binding site for minus-strand DNA synthesis followed the 5' LTR. The U3 region contained putative transcriptional promoters, "CAT" box and "TATA" box, but they had peculiar sequences (C-C-A-A-C-T and G-A-T-A-A-A-T). The U3 region also contained a potential enhancer element, whose sequence partially resembled those of other viral and cellular, especially of immunoglobulin, enhancers. The most striking structural feature of the LTR was an exceptionally long R region (228 bp), which separated a poly(A) addition signal (A-A-T-A-A-A) from a poly(A) site as far apart as 260 bp. The long R region was suggested to form a large stable hairpin structure on a nascent RNA chain, making the two transcription termination signals close together and thus ensuring normal termination of the chain. This structural feature of the bovine leukemia virus LTR was analogous to that of human T-cell leukemia virus LTR and, in fact, slight sequence homology (at most 50%) was observed between the R regions of these two retroviruses, indicating their evolutionary relationship. The unique structural feature of bovine leukemia virus and human T-cell leukemia virus LTRs may thus bear some relation to the biological features commonly shared by these retroviruses.

Human immunodeficiency virus (HIV), the causative agent of AIDS, infects and kills lymphoid cells bearing the CD4 antigen. In an infected cell, a number of cellular as well as HIV-encoded gene products determine the levels of viral gene expression and HIV replication. Efficient HIV-replication occurs in activated T cells. Utilizing transient expression assays, we show that gene expression directed by the HIV long terminal repeat (LTR) increases in response to T-cell activation signals. The effects of T-cell activation and of the HIV-encoded trans-activator (TAT) are multiplicative. Analysis of mutations and deletions in the HIV LTR reveals that the region responding to T-cell activation signals is located at positions -105 to -80. These sequences are composed of two direct repeats, which are homologous to the core transcriptional enhancer elements in the simian virus 40 genome. Our studies reveal that these elements function as the HIV enhancer. By acting directly on the HIV LTR, T-cell activation may play an important role in HIV gene expression and in the activation of latent HIV.

We have isolated and sequenced a clone from a rainbow trout (Salmo gairdneri) genomic library that carries a gene encoding a fish growth hormone (GH). This gene spans a region of approximately equal to 4 kilobases, nearly twice that of mammalian GH genes. The trout GH gene is comprised of six exons, in contrast with five exons in mammals. The additional intron in the fish gene interrupts translated regions that are analogous to the last exon of its mammalian counterpart. In addition, the alleged internally repeating sequence in mammalian GH, prolactin (Prl), or placental lactogen (PL) is not observed in the predicted polypeptide sequence of fish GH. Direct repeats that flank exons I, III, and V of the mammalian GH, Prl, and PL genes are absent in the fish GH gene. These findings indicate that the rainbow trout GH gene structure does not support the current hypothesis that internally repeated regions in GH, Prl, and PL arose from a small primordial gene.

Crown gall disease results from the insertion of a segment of the Agrobacterium Ti plasmid, called T-DNA, into host plant nuclear DNA. We have subjected to sequence analysis the border regions of pTi T37 (ends of T-DNA) and one left T-DNA/plant DNA border fragment isolated from BT37 tobacco teratoma by molecular cloning. These sequence studies, taken together with published sequence of a right T-DNA/plant DNA border fragment, allowed us to identify the positions of left and right borders at the DNA sequence level. Comparison of left and right border regions of the Ti plasmid revealed a “core” direct repeat of 13 of 14 bases (12 contiguous) precisely at the borders of T-DNA. An extended repeat of 21 of 25 bases overlaps this core repeat. T-DNA on the Ti plasmid exhibits no longer direct or inverted repeats in the border regions, based on Southern hybridization studies. The physical structure of T-DNA differs from that of known prokaryotic and eukaryotic transposable elements but bears a structural resemblance to the prophage of bacteriophage λ.

A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps.

Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.

The photosynthetic cyanobacterium Synechococcus R2 is transformed by chimeric donor molecules lacking a functional replication origin but containing a region of homology to the recipient chromosome. These integrating donor molecules consist of a fragment of Synechococcus R2 chromosomal DNA cloned in the Escherichia coli vector pBR322 and interrupted by a piece of foreign DNA. During integration, this interrupting DNA is often lost by nonreciprocal exchange between homologous regions of donor and recipient. When transformed with donor molecules containing in vitro -generated double-stranded gaps or deletions as large as 20 kilobase pairs in the fragment homologous to the recipient chromosome, Synechococcus R2 can repair these lesions by using recipient information. Chromosomal DNA of the resulting transformants contains direct repeats of the recipient copy on either side of integrated pBR322 DNA. Homologous recombination between these repeats generates a circular molecule that can be recovered by transformation to E. coli . Plasmids recovered in E. coli contain the entire copy of information initially present in the region of the Synechococcus recipient corresponding to the donor gap or deletion. We suggest applications of this mechanism for cloning of genes identified by transposon mutagenesis.

Replicative transposition is signaled by the formation of cointegrates in which donor and target replicons are joined by direct repeats of a transposable element. Elements not generating such cointegrates may move by a conservative breaking and joining process. The IS50 elements forming the terminal repeats of Tn5 [which carries the determinant for kanamycin resistance (Kanr)] contain genes and sites necessary for transposition and mediate the movement of any DNA segment they bracket. To determine if IS50 generates cointegrates, the products of transposition from pBR322::Tn5 plasmids to an F factor in recA-Escherichia coli were examined. With monomeric pBR322::Tn5 plasmids, transposition of Kanr (from Tn5) was generally not accompanied by movement of the determinant for ampicillin resistance (Ampr) (from the pBR322 vector). With dimeric pBR322::Tn5 plasmids, by contrast, half of the transpositions of kanr were accompanied by transposition of ampr. Restriction endonuclease analyses indicated that these F-Kanr Ampr chimeras contained inserts of a single copy of the pBR322 vector sequence bracketed by one Tn5 element and one IS50 element or by a pair of Tn5 elements. None of 79 chimeras tested was a true cointegrate. Because IS50 seems to move only a segment of the donor replicon it is proposed that IS50 transposition is conservative.

The regions 5' proximal to many yeast tRNA genes exhibit a high frequency of DNA sequence polymorphisms. DNA sequence analysis of polymorphic variants of SUQ5, a tRNA Ser UCA gene, and SUP2, a tRNA Tyr gene, shows that in each case one sequence variant of the tRNA gene is 346 base pairs longer than the other. The longer variants appear to have arisen from the shorter ones by the insertion of nearly identical copies of a 341-base pair sigma element into a site 16 base pairs upstream from the 5' ends of the tRNA-coding regions. The sequences of the two copies of the sigma element differ at only five positions. The element has a number of properties that are typical of many transposable elements: (i) there is a perfect eight-base-pair inverted repeat at its ends, (ii) these ends are flanked by a five-base-pair direct repeat of a sequence that occurs only once in the target DNA, (iii) there are approximately 20 copies of the element in the yeast genome, and (iv) there is considerable strain-to-strain variation in the sizes of the restriction fragments on which these copies lie. The presence of the sigma element has no gross effect on the phenotype of a SUP2 ochre suppressor. Analysis of the SUQ5 and SUP2 sequences favors the hypothesis that sigma is a transposable element with a novel type of insertion specificity, which is primarily based on the presence of a tRNA-coding region a fixed distance from the insertion site, rather than on the immediate target sequences.

A gene coding for proteinase inhibitor I, whose expression is induced in tomato leaves (Lycopersicon esculentum L. var. Bonny Best) in response to wounding or insect attacks, was isolated from a genomic library and characterized. The nucleotide sequence revealed that the gene is complete and encodes the sequence of an inhibitor I cDNA that was previously isolated from a cDNA library prepared from wound-induced mRNA from tomato leaves. This gene is located 13.1 kilobase pairs (kbp) upstream from an inhibitor II gene. The wound-inducible gene is interrupted by two intervening sequences of 445 and 404 bp, situated within the codons of amino acids 17 and 47, respectively, of the open reading frame. In addition to the presence of putative regulatory sequences, TATAAA and CCACT, two copies of an imperfect direct repeat approximately 100 bp long were identified in the 5'-flanking region. Phylogenetic comparisons of wound-inducible inhibitor I genes within the genomes of various Lycopersicon species revealed that the repeat is found in seven ancestral species of tomato.

We have determined the nucleotide sequences of the long terminal repeats of the transposable element gypsy from the cloned mutant alleles sc1, bx3, and bx34e. These mutations are suppressible by the suppressor of Hairy-wing, su(Hw). The long terminal repeats are 482 base pairs long and are highly conserved. In each case, gypsy is inserted into the sequence T-A-C-A-T-A and generates a duplication of the sequence T-A-C-A. This was verified by sequencing an empty site in the wild-type bx gene. Consideration of the sequence of the long terminal repeats and their surroundings limits the possible explanations for the mechanism of mutation by these gypsy insertions and for their suppression by su(Hw).

We describe a 5- to 6-kilobase-pair repetitive family in human DNA. One member of this family is linked to the beta-globin gene cluster and is close to the 3' breakpoints of three different naturally occurring deletions involving this gene cluster. Sequence analysis indicates that this element includes terminal direct repeats of 415 base pairs that exhibit the features of long terminal repeats (LTRs) of retroviruses. A potential histidine tRNA primer binding site occurs just 3' to the 5' direct repeat. This retrovirus-like element interrupts a member of the Kpn I family of repeated DNA and is bracketed by a 5-base-pair directly repeated sequence. When attempts are made to clone the element in bacteriophage, homologous recombination between the LTR-like sequences is very frequently observed. Copy number estimates by two methods indicate that the element is repeated 800-1000 times in the human genome. We term this Homo sapiens family of retrovirus-like elements having a histidine tRNA primer binding site the hsRTVL-H family.

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes and cloned into the HindIII site of lambda vector Charon 7. Fifty-six recombinants from this spc DNA library were analyzed. R repeats, which were originally found near immunoglobulin genes, were enriched in spc DNA clones relative to their representation in the chromosome. In one clone, the R sequence was linked to Bam and MIF sequences and the contiguous arrangement was truncated from both ends. In another clone, composite Bam/R and R repeats existed as a pair in inverted repeat orientation. Truncation occurred from the 5' side without affecting the 3' ends. In both clones, short direct repeats flanked the repeated sequences. The possible role of R sequences in transposition and circular formation is discussed.

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.