Chromosome Walking Experiments Research Articles

Chlamydomonas Flagellar Outer Row Dynein Assembly Protein Oda7 Interacts with Both Outer Row and I1 Inner Row Dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Integration of chicken genomic resources to enable whole-genome sequencing

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HindIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.

A YAC contig map of Arabidopsis thaliana chromosome 3

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low-copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large-scale DNA sequencing of chromosome 3.

A Yeast Artificial Chromosome Contig That Spans the RB1-D13S31 Interval on Human Chromosome 13 and Encompasses the Frequently Deleted Region in B-cell Chronic Lymphocytic Leukemia

Abnormalities involving chromosome 13 have been reported as the only cytogenetic change in B-cell chronic lymphocytic leukemia (BCLL). Deletions are the most common cytogenetic abnormality and always involve 13q14, but when translocations are seen, the consistent breakpoint is always in 13q14. It is now established that deletions, distal to the RB1 gene in 13q14, are invariably associated with these translocations. We have recently described the smallest such deletion from a series of rearrangements from these tumors isolated in somatic cell hybrids, which spans approximately 1 Mb. In this report, we present the results of a series of a chromosome walking experiments using YACs and have been able to span this small deletion, which must contain the gene that is frequently deleted in BCLL. Four probes from 13q14 (RBI-mgg15-D13S25-D13S31) were used to isolate corresponding YACs for each of the markers. The chromosomal location of these YACs was verified using FISH, which also demonstrated their nonchimeric nature. Vectorette end rescue was then used to demonstrate the overlap of the YACs and to isolate new clones to complete the contig. The extremes of the contig were shown to cross the chromosome 13 translocation breakpoints isolated in somatic cell hybrids that carry the derivatives of chromosome 13 involved in the smallest BCLL deletion. This YAC contig covers the entire deletion and will prove a valuable resource to begin isolating genes from this region. In addition, we have isolated YACs corresponding to the RB1 locus, which extends the contig over a 3.8-cM distance on the chromosome.

A cosmid with a HyR marker for fungal library construction and screening.

The construction of a double-cos-site cosmid vector, pMOcosX, for use in making filamentous fungal genomic DNA libraries, is described. The vector has features that allow for selection of clones introduced into fungi by transformation and for efficient chromosome walking experiments. These features include (i) two cos sites allowing for easy construction of libraries without requiring size selection of insert DNA; (ii) an XhoI site for insertion of Sau3AI or MboI partially digested genomic DNA inserts that allows usage of a half-site fill-in method which minimizes the possibility of producing clones containing chimeric inserts; (iii) a bacterial hygromycin phosphotransferase-encoding gene fused to a modified cpc-1 promoter of Neurospora crassa for direct selection of cosmid clones upon introduction into fungal cells; and (iv) T7 and T3 bacteriophage promoters and EcoRI, NotI and BamHI restriction sites flanking the cloning site that allow for synthesis of, or isolation of, end-specific probes for chromosome walking. The combination of features in this vector allows for the easy construction and use of high-quality fungal DNA libraries from small amounts of genomic DNA.

The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening

ABSTRACTIn the years since we last reviewed the use of mutants to study tomato fruit ripening (Grierson et al. 1987), considerable information has been gained by the cloning, sequencing and identification of many mRNAs implicated in this developmental process. Genes involved in cell wall degradation, colour change and ethylene synthesis have been cloned, and antisense techniques have been developed and used to produce genetically engineered mutant fruit deficient in these aspects of ripening (see Gray et al. 1992). Recently, a previously cloned ripening gene has been used to complement a naturally occurring fruit colour mutant, yellow flesh (Fray & Grierson 1993a), and a ripening impaired mutant, ripening inhibitor, has been used to identify several new ripening‐related mRNAs (Picton et al. 1993b). The chromosomal region bearing the ripening inhibitor mutation has been subjected to high‐resolution mapping (Churchill, Giovannoni & Tanksley 1993) and chromosome walking experiments are in progress to identify this gene.

Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA.

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.

Chromosome walking with YAC clones in Arabidopsis: isolation of 1700 kb of contiguous DNA on chromosome 5, including a 300 kb region containing the flowering-time gene CO

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-length. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%-2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.

Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly-sheared Arabidopsis thaliana DNA

We describe the construction of a yeast artificial chromosome (YAC) library from the Arabidopsis thaliana genome. Randomly sheared high molecular weight source DNA was extracted from frozen, ground leaf tissue and blunt-end-ligated to the vector pYAC3. By size-fractionating the ligation products, we achieved an average clone size of 150 kb. Approximately 6% of the YACs contained inserts from the chloroplast genome. We screened clones equivalent to greater than four A. thaliana haploid nuclear genomes and isolated YACs homologous to five single-copy-sequence probes. The library should be useful for chromosome walking and genome mapping experiments. In addition, the approach used for its construction should be applicable to other higher plant species.

Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes

Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes.

Characterization of a four-member psbA gene family from the cyanobacterium Anabaena PCC 7120

The cyanobacterium Anabaena PCC 7120 contains a multigene family that encodes the D1 polypeptide of Photosystem II. This family consists of four members denoted psbAI-IV that are each unique but highly homologous. psbAII, III and IV are more closely related to each other than to psbAI. These three copies encode identical polypeptides that differ from the psbAI product by 21 amino acids. The transcription initiation site for psbAI has been mapped to 64-65 nucleotides upstream from the coding region. Primer extension assays performed with an oligonucleotide specific for psbAII, III and IV transcripts suggest that one or more of these genes is also expressed. Genomic mapping and chromosome walking experiments demonstrate that none of the four psbA copies is within 20 kbp of another member of the gene family.

Recombinant inbred strain and interspecific backcross analysis of molecular markers flanking the murine agouti coat color locus.

Recombinant inbred strain and interspecific backcross mice were used to create a molecular genetic linkage map of the distal portion of mouse chromosome 2. The orientation and distance of the Ada, Emv-13, Emv-15, Hck-1, Il-1a, Pck-1, Psp, Src-1 and Svp-1 loci from the beta 2-microglobulin locus and the agouti locus were established. Our mapping results have provided the identification of molecular markers both proximal and distal to the agouti locus. The recombinants obtained provide valuable resources for determining the direction of chromosome walking experiments designed to clone sequences at the agouti locus. Comparisons between the mouse and human genome maps suggest that the human homolog of the agouti locus resides on human chromosome 20q. Three loci not present on mouse chromosome 2 were also identified and were provisionally named Psp-2, Hck-2 and Hck-3. The Psp-2 locus maps to mouse chromosome 14. The Hck-2 locus maps near the centromere of mouse chromosome 4 and may identify the Lyn locus. The Hck-3 locus maps near the distal end of mouse chromosome 4 and may identify the Lck locus.

Storage of unamplified phage libraries on nylon filters.

Publisher Summary The filter protocol has been successfully used for the storage of both genomic DNA and complementary DNA (cDNA) libraries. Positive phage are recovered from the filter at the same efficiency as agarose plugs taken from the same area of the primary plates. The stability of the amplified libraries made by scraping off the top agarose from the plates after plaque lifts have been made is also very good. A potential limitation of the protocol is the number of times the replica filters can be screened before the loss of signal is apparent. Stripping of probe signal from the filters before rehybridizing with other probes is not recommended as positive signals from previous screens can be easily identified and so disregarded. For chromosome walking experiments, the ability to see positive phage from previous screens is advantageous as it avoids wasted effort on characterizing clones already isolated at the previous walking step. The ability to make both an amplified and unamplified version of the same plated library removes the dilemma that has faced many workers in the past––whether to screen the primary library directly or to amplify it first and run the risk of missing an underrepresented clone.

MIC2: a human pseudoautosomal gene.

MIC2 and XGR are the only known pseudoautosomal genes in man. MIC2 encodes the 12E7 antigen, a human cell-surface molecule of unknown function. XGR regulates, in cis, the expression of the XG and MIC2 genes. DNA probes derived from the MIC2 locus have been used in the construction of a meiotic map of the pseudoautosomal region and a long range restriction map into the X- and Y-specific chromosome domains. MIC2 is the most proximal marker in the pseudoautosomal region and recombination between the sex chromsomes only rarely includes the MIC2 locus. Our long-range restriction maps and chromosome walking experiments have localized the pseudoautosomal boundary within 40 kilobases adjacent to the 3' end of the MIC2 gene. The same maps have been used to predict the chromosomal location of TDF.

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.

Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (≤80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.

Genetic organization of the agouti region of the mouse.

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.