Advances in maize genomics: the emergence of positional cloning

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.     

Toward positional cloning of the curly tail gene

BACKGROUND: The curly tail (ct) mutant mouse is one of the best-studied mouse models of spina bifida. The ct mutation has been localized to distal chromosome 4 in two independent studies and was recently postulated to be in the Grhl-3 gene. METHODS: A recombinant BALB/c-ct strain was generated and used to precisely map the ct gene. RESULTS: We report the absence of gross chromosomal abnormalities and the precise mapping of the ct gene to a 3-Mb region at 135 Mb (66 cM) from the centromere, closely linked to the polymorphic microsatellite marker D4Mit148. Candidate genes, Idb3, Wnt4, Cdc42, and perlecan, all localized in the critical region, were studied by sequence and expression analyses. Our data indicate that these genes in all probability do not account for the ct phenotype. In addition, our expression data do not provide strong evidence that Grhl-3 is indeed the ct gene. CONCLUSIONS: The ct gene has not yet been identified. A total of 29 candidate genes remain present in the critical region. Refined mapping studies need to be performed to further narrow the region and additional candidate genes need to be examined. Supplementary material for this article can be found on the Birth Defects Research (Part A) website (http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2005/73/tables_S3-S6.doc).     

RAPD-based screening of genomic libraries for positional cloning.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.     

Evidence for purifying selection acting on silent sites in BRCA1

In mammals, it is usually assumed that selection cannot be strong enough to act on nucleotide mutations that do not cause a change at the protein level (i.e. 'silent' or 'synonymous' mutations). Here we report the results of a molecular evolutionary analysis of BRCA1. We find a repeatable pronounced peak in the ratio of nonsynonymous to synonymous substitutions between codons 200-300. Unusually, this peak is caused by a plummet in the silent-site rate of evolution. The most parsimonious interpretation of these data is that purifying selection is acting on silent sites.     

Reconstructing the history of selection during homoploid hybrid speciation

This study aims to identify selection pressures during the historical process of homoploid hybrid speciation in three Helianthus (sunflower) hybrid species. If selection against intrinsic genetic incompatibilities (fertility selection) or for important morphological/ecological traits (phenotypic selection) were important in hybrid speciation, we would expect this selection to have influenced the parentage of molecular markers or chromosomal segments in the hybrid species' genomes. To infer past selection, we compared the parentage of molecular markers in high-density maps of the three hybrid species with predicted marker parentage from an analysis of fertility selection in artificial hybrids and from the directions of quantitative trait loci effects with respect to the phenotypes of the hybrid species. Multiple logistic regression models were consistent with both fertility and phenotypic selection in all three species. To further investigate traits under selection, we used a permutation test to determine whether marker parentage predicted from groups of functionally related traits differed from neutral expectations. Our results suggest that trait groups associated with ecological divergence were under selection during hybrid speciation. This study presents a new method to test for selection and supports earlier claims that fertility selection and phenotypic selection on ecologically relevant traits have operated simultaneously during sunflower hybrid speciation.     

Advances in the positional cloning of nodulation genes in soybean

The effect of liming on the decomposition of Norway spruce needle litter was studied in 40–60-year-old Norway spruce stands. Finely-ground limestone had been spread about 30 years ago at a dose of 2 t ha^–1 and reliming was carried out about 20 yr later at a dose of 4 t ha^–1. Needle litter was collected from both control and limed plots, and it was placed in litter bags in the middle of the humus layer of the plot from which they originated, and similarly to the other plot in May. Litter bags were sampled after 4, 12 and 16 months. The site of origin of the needle litter, whether from control plot or from limed plot, affected mainly the early stages of decomposition. Initially the effect of liming was seen as decreased concentration of water soluble material and then, during decomposition, as decreased mass loss and decreased degradation of lignin, and increased C/N ratio. The incubation site, whether the control or the limed plot, did not affect decomposition significantly.Decomposition of Scots pine needles in a young Scots pine plantation was also studied. The treatments were: 2 t ha^–1 of finely-ground limestone and 2.5 t ha^–1 of bark ash spread 8 months before this study. The treatments did not affect decomposition much, but some stimulation of the treatments on decomposition was observed. Compared to spruce needles, the C/N ratio of pine seedles was lower, they contained less lignin and more water soluble material, and decomposed faster in the first summer.     

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.     

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.     

Limits of resolution of genetic linkage studies: implications for the positional cloning of human disease genes.

Positional cloning studies to identify disease genes are being carried out for many human genetic diseases. Such studies often include a genome-scan linkage analysis to identify the rough chromosomal location of a disease gene, fine structure genetic mapping to define and narrow the chromosomal interval in which the disease gene may be located, and physical mapping and gene identification in the genetically defined interval to clone the disease gene. During the planning of a positional cloning study, it is important to know that, if linkage is found, the genetic interval identified is likely to be sufficiently narrow to be dissected efficiently by methods of physical mapping and gene identification. Thus, we wish to know the limits of resolution of a genetic linkage study. In this paper, I determine for Mendelian diseases the distributions and moments of three measures of linkage resolution: (1) in a set of N chromosomes, the distance between the nearest crossovers that flank a disease locus, (2) the distance between the nearest genetic markers that flank the pair of flanking crossovers after a genome scan, and (3) the distance between the nearest flanking markers after additional randomly placed markers are generated and typed in an identified interval. These results provide explicit sample-size guidelines for future positional cloning studies of Mendelian diseases and make possible a more objective evaluation of whether a proposed positional cloning study is likely to be successful. I also briefly discuss the more difficult problem of linkage resolution for complex genetic diseases.     

ColE1 vectors for direct selection of cells carrying a hybrid plasmid

pKY2289, a ColE1::Tn3 derivative, useful for direct selection of cells carrying a hybrid plasmid, was deleted of its mobility functions and parts of the transposon including the left inverted repeat. This deletion mutant, named pKY2700 expresses low levels of colicin E1 synthesis even in recA cells. A nitrosoguanidine mutant of pKY2289 which shows a high level of constitutive colicin E1 synthesis was also deleted of the same sequences as pKY2700. The second plasmid, named pKY2800, has the same molecular weight (3.8 megadaltons) and almost the same structure as pKY2700, but produces colicin E1 at much higher levels and has a copy number 10 times higher. pKY2800 requires no colicin E1 induction for the direct selection of hybrid clones, while pKY2700 requires mitonmycin C at a concentration of 10 ng per ml. These two colE1 derivatives are useful as safe and convenient vectors for cloning DNA fragments at the cleavage sites of EcoRI, XmaI, and SstII of plasmid.     

X-chromosome inactivation: X marks the spot for BRCA1

X-chromosome inactivation equalizes the dosage of X-linked genes in XX females with that in XY males. Recent findings reveal that the BRCA1 breast cancer susceptibility gene has an important function in this epigenetic phenomenon.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

Untangling the crosstalk between BRCA1 and R-loops during DNA repair

R-loops are RNA:DNA hybrids assembled during biological processes but are also linked to genetic instability when formed out of their natural context. Emerging evidence suggests that the repair of DNA double-strand breaks requires the formation of a transient R-loop, which eventually must be removed to guarantee a correct repair process. The multifaceted BRCA1 protein has been shown to be recruited at this specific break-induced R-loop, and it facilitates mechanisms in order to regulate R-loop removal. In this review, we discuss the different potential roles of BRCA1 in R-loop homeostasis during DNA repair and how these processes ensure faithful DSB repair.     

Synthesis of pyrazole-based hybrid molecules: search for potent multidrug resistance modulators

The hybrid molecules have been designed on the basis of the structural features of pyrazole-based drugs and MDR modulator propafenone. A simple synthetic strategy and solvent-based regioselectivity have been used for the synthesis of newly designed molecules and they are evaluated for their interactions with P-glycoprotein (P-gp). Some of the molecules show considerable interactions with P-gp and compounds 15, 28 and 40 could be the potential candidates for their use as MDR modulators.     

Practical aspects of donor cell selection for nuclear cloning

Choosing the right nuclear donor is the most critical decision in cloning by nuclear transfer (NT), or nuclear cloning, because the cloned animal will be a genetic copy of the donor cell genome used for NT. Both donor cell type and cell cycle stage are important methodological parameters and influence nuclear cloning efficiency. Cloning, however, is a multi-step procedure and the exact contribution of the nuclear donor to overall cloning success must be determined in comparative studies. This requires strict standardization of isolation, purification, and culture protocols, and application of stringent identification criteria in order to obtain a homogenous donor cell population. In all these respects, the standards in the cloning field are currently poor. The aim of this review is to provide a brief guideline for the major practical aspects of donor cell selection, cell cycle synchronization and preparation for NT.