Single-Step Conversion of P1 and P1 Artificial Chromosome Clones into Yeast Artificial Chromosomes

Genomics ◽  
2000 ◽  
Vol 68 (1) ◽  
pp. 106-110 ◽  
Author(s):  
Parvoneh Poorkaj ◽  
Kenneth R. Peterson ◽  
Gerard D. Schellenberg
Keyword(s):  
Artificial Chromosome ◽  
Single Step ◽  
Artificial Chromosomes ◽  
Yeast Artificial Chromosomes

Arabidopsis YAC restriction mapping

Genome ◽  
1998 ◽  
Vol 41 (6) ◽  
pp. 806-817
Author(s):  
Shaun M Morroll ◽  
Zoe A Wilson
Keyword(s):  
Positional Cloning ◽  
Floral Development ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Genomic Region ◽  
Restriction Mapping ◽  
Artificial Chromosomes ◽  
Number Of Genes ◽  
Yeast Artificial Chromosomes ◽  
Large Genomic Region

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development.

scholarly journals Meiotic recombination on artificial chromosomes in yeast.

Genetics ◽  
1992 ◽  
Vol 131 (3) ◽  
pp. 541-550 ◽  
Author(s):  
L O Ross ◽  
D Treco ◽  
A Nicolas ◽  
J W Szostak ◽  
D Dawson
Keyword(s):  
Meiotic Recombination ◽  
Artificial Chromosome ◽  
Artificial Chromosomes ◽  
Crossover Behavior ◽  
Yeast Chromosome ◽  
Low Levels ◽  
Chromosome Iii ◽  
The Right ◽  
Yeast Artificial Chromosomes ◽  
Derivatives Of

Abstract We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.

scholarly journals Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations

1990 ◽  
Vol 56 (2-3) ◽  
pp. 203-208 ◽  
Author(s):  
Zoia Larin ◽  
Hans Lehrach
Keyword(s):  
Mouse Genome ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Artificial Chromosome ◽  
Molecular Study ◽  
Insert Size ◽  
Artificial Chromosomes ◽  
Partial Digestion ◽  
Genomic Regions ◽  
Yeast Artificial Chromosomes

SummaryMammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NoiI and BssHII, and the detection of gene loci from the H-2 complex, the t–complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t–complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.

scholarly journals ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes

2020 ◽  
Vol 4 (2) ◽  
pp. e202000836
Author(s):  
Arnaud Rondelet ◽  
Andrei Pozniakovsky ◽  
Devika Namboodiri ◽  
Richard Cardoso da Silva ◽  
Divya Singh ◽  
...  
Keyword(s):  
Protein Function ◽  
High Efficiency ◽  
Marker Gene ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Single Step ◽  
Step Method ◽  
Mutation Site ◽  
Endogenous Gene ◽  
Artificial Chromosomes

Bacterial artificial chromosome (BAC)–based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

scholarly journals Xist Yeast Artificial Chromosome Transgenes Function as X-Inactivation Centers Only in Multicopy Arrays and Not as Single Copies

1999 ◽  
Vol 19 (4) ◽  
pp. 3156-3166 ◽  
Author(s):  
Edith Heard ◽  
Fabien Mongelard ◽  
Danielle Arnaud ◽  
Philip Avner
Keyword(s):  
X Chromosome ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Diploid Cell ◽  
Single Copy ◽  
X Inactivation ◽  
Artificial Chromosomes ◽  
Autonomous Function ◽  
Inactivation Center ◽  
Yeast Artificial Chromosomes

ABSTRACT X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation incis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

Murine Albino-Deletion Complex: High-Resolution Microsatellite Map and Genetically Anchored YAC Framework Map

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 787-799
Author(s):  
Brad A Rikke ◽  
Dabney K Johnson ◽  
Thomas E Johnson
Keyword(s):  
Positional Cloning ◽  
Interspecific Backcross ◽  
Genetic Distances ◽  
Sequence Length ◽  
Recombination Rates ◽  
Oak Ridge ◽  
Artificial Chromosomes ◽  
Microsatellite Map ◽  
Specific Locus ◽  
Yeast Artificial Chromosomes

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6–11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region suitable for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3–0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deleted region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

A Novel Ty1-Mediated Fragmentation Method for Native and Artificial Yeast Chromosomes Reveals That the Mouse Steel Gene is a Hotspot for Ty1 Integration

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 673-683
Author(s):  
Jacob Z Dalgaard ◽  
Mukti Banerjee ◽  
M Joan Curcio
Keyword(s):  
Transcription Unit ◽  
Yeast Genome ◽  
Physical Analysis ◽  
Single Chromosome ◽  
Artificial Chromosomes ◽  
Unique Site ◽  
High Fraction ◽  
A Site ◽  
Yeast Artificial Chromosomes ◽  
Random Insertion

Abstract We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24 retrotransposon insertions into two different mousederived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into aYAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.

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