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.     

A simple method for cloning leishmanial promastigotes

A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3 X 10(3) promastigotes per ml. Hanging-drop preparations made from 0.2-0.4 microliter volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22 degrees C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species of Leishmania, with up to 100 percent of the cloned organisms growing.     

Positive-selection cloning vehicle useful for overproduction of hybrid proteins.

Plasmid pSCC31 contains the EcoRI endonuclease gene downstream from lambda pL. It does not yield transformants upon introduction into Escherichia coli unless the structural integrity of the endonuclease is destroyed. This makes it useful as a positive-selection cloning vehicle which can be employed for regulated overproduction of hybrid proteins.     

A simple formula for obtaining markedly improved mutation rate estimates

In previous work by M. E. Jones and colleagues, it was shown that mutation rate estimates can be improved and corresponding confidence intervals tightened by following a very easy modification of the standard fluctuation assay: cultures are grown to a larger-than-usual final density, and mutants are screened for in only a fraction of the culture. Surprisingly, this very promising development has received limited attention, perhaps because there has been no efficient way to generate the predicted mutant distribution to obtain non-moment-based estimates of the mutation rate. Here, the improved fluctuation assay discovered by Jones and colleagues is made amenable to quantile-based, likelihood, and other Bayesian methods by a simple recursion formula that efficiently generates the entire mutant distribution after growth and dilution. This formula makes possible a further protocol improvement: grow cultures as large as is experimentally possible and severely dilute before plating to obtain easily countable numbers of mutants. A preliminary look at likelihood surfaces suggests that this easy protocol adjustment gives markedly improved mutation rate estimates and confidence intervals.     

New type of linker useful for cloning experiments

A new class of linker oligodeoxynucleotide sequences is described which allows the original sequence of a foreign DNA to be restored after cloning. In the standard linker approach the terminal base pairs of the linker-sequences are irreversibly attached to the cloned DNA, thus altering the genetic information. The use of the new type of linker is demonstrated by the transformation of the unique Pvu II site in pBR322 into Bam HI site using the ISO ('in-site-out') linker d(GATCCGGATC). Possible further applications of these linker sequences are described.     

A simple,efficient, standard-dilution method for cloning hybridomas

Summary A simple standard-dilution method which obviates routine use of viable cell counts, feeder layers and time-consuming scale-up procedures is described. This method can be used to clone monoclonal antibody-producing hybridomas 9–14 days post-fusion. Each cloning cycle takes 5 min. Approximately 60% of the positive, monoclonal antibody-producing hybridomas were successfully cloned and established.     

Long-range walking techniques in positional cloning strategies

Conclusion The past several years have seen an explosive growth in our understanding of the organization and structure of mammalian genomes, and refinements of existing techniques for genetic analysis, physical mapping, and large-fragment cloning techniques may well be enough to continue the momentum of that explosion for some time to come. Although refinement of existing techniques will certainly be necessary, the development of new and better cloning techniques may, perhaps, no longer be our most urgent need. The most important challenge that we face at present may in fact be that of finding efficient ways to share existing resources and information rapidly and equitably throughout the scientific community so that progress can continue unimpeded, and to catalog, correlate, and interpret the wealth of new data that is so rapidly accumulating.New strategies aimed at whole-genome mapping (Coulson et al. 1986, 1988; Michiels et al. 1987; Brenner and Livak 1989; Carrano et al. 1989; Lehrach et al. 1991) and sequencing (Church and Keifer-Higgins 1988; Bains and Smith 1988; Drmanac et al. 1989; Strzoska et al. 1991) may someday make the current method of long-range walking and physical mapping nearly passe. For example, since most of the relatively small nematode genome is now stored as ordered sets of cosmid and YAC clones (Coulson et al. 1986, 1988), a walk between a mapped marker and an uncloned gene can be accomplished rapidly, through a request for the appropriate series of clones from the ordered library. Vigorous drives by many laboratories to produce ordered clone libraries for murine and human chromosomes (Lehrach et al. 1991) may transform the process of cloning mammalian genes into a relatively trivial matter within the foreseeable future. The remarkable number of positional-cloning successes that have been reported in recent years may indicate that most of the best-defined, simply inherited mouse mutations and human hereditary disorders will have already been cloned by that time. When that is accomplished, the true challenging task will just begin: we must learn to decipher the complex biological programs encoded by our large and ever-growing storehouse of cloned, mapped and sequenced genes, before we can begin to understand what might be held in the vast silent mass of mammalian genomes. Offprint requests to: L. Stubbs     

A simple and rapid method for cloning insect vitellogenin cDNAs

We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A)+ RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7-0.9 kb, representing the 3' portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene-specific primers corresponding to the sequences near the 5'-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5' portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes.     

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.     

Methods for finding genes a major rate-limiting step in positional cloning

Identification of transcribed sequences from within genomic regions has been a major rate-limiting step in the pursuit of genes involved in many human genetic diseases. Early efforts focused primarily on screening of cDNA libraries, identification of evolutionary conserved sequences, and northern blot hybridization. In recent years, several innovative techniques for gene identification have been devised. These techniques expand the size of the genomic region capable of being scanned for genes, while also allowing detection of genes regardless of their expression patterns. This article reviews several new and older techniques and discusses the advantages and limitations of each.     

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).     

定位克隆分离植物基因的研究进展

定位克隆是分离基因的有效方法。本文对定位克隆的原理和方法进行了简要介绍,概括了定位克隆的分离植物基因上的研究进展,分析了其局限性和解决方法,并对定位克隆的应用前景做出展望。     

A simple method for cloning and replica plating of mammalian cells using multicellular spheroids.

V 79/4 Chinese hamster cells or HeLa cells grow in Eagle's MEM supplemented with 25 microgram/ml dextran sulphate to form clonal multicellular spheroids. These cell clones, consisting of 5-10(2) cells, are easy to separate, to transfer from one culture vessel into another and grow as normal monolayer colonies on Dederon cloth circles after subculture in Eagle's MEM without dextran sulphate. A simple replica technique is described by which 500 clones can be transfered onto at least 3 replica cloth circles, 10 cm in diameter, with a replica plating efficiency of approximately 100%.