Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered “single-copy” (or better “two-copy”) sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y′ repeat, while 15 end with the 800-nt “X2” repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

Stable transfer of intact high molecular weight DNA into plant chromosomes.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

Synaptonemal complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes.

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.

Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones.

A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

The genome of Caenorhabditis elegans.

The physical map of the 100-Mb Caenorhabditis elegans genome consists of 17,500 cosmids and 3500 yeast artificial chromosomes (YACs). A total of 22.5 Mb has been sequenced, with the remainder expected by 1998. A further 15.5 Mb of unfinished sequence is freely available online: because the areas sequenced so far are relatively gene rich, about half the 13,000 genes can now be scanned. More than a quarter of the genes are represented by expressed sequence tags (ESTs). All information pertaining to the genome is publicly available in the ACeDB data base.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Preferential isolation of DNA fragments associated with CpG islands.

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.