Yeast artificial chromosome libraries containing large inserts from mouse and human DNA.

Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.

Download Full-text

Structure of the Chromosome VII Centromere Region in Neurospora crassa: Degenerate Transposons and Simple Repeats

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5465 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5465-5477 ◽

Cited By ~ 69

Author(s):

Edward B. Cambareri ◽

Rafael Aisner ◽

John Carbon

Keyword(s):

Neurospora Crassa ◽

Yeast Artificial Chromosome ◽

Fragment Length Polymorphism ◽

Artificial Chromosome ◽

Polymorphism Analysis ◽

Dna Repeats ◽

Centromere Region ◽

Simple Repeats ◽

Restriction Fragment Length ◽

Repeat Induced Point Mutation

ABSTRACT DNA from the centromere region of linkage group (LG) VII ofNeurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical ofNeurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (∼16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3′ end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobilecopia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to theTy3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcenelements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in theDrosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.

Download Full-text

Entire maize chloroplast genome is stably maintained in a yeast artificial chromosome

Plant Molecular Biology ◽

10.1007/bf00040631 ◽

1991 ◽

Vol 17 (3) ◽

pp. 361-369 ◽

Cited By ~ 6

Author(s):

Manju Gupta ◽

Brad Hoo

Keyword(s):

Chloroplast Genome ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Maize Chloroplast

Download Full-text

Chromosomal localization of human genes for arylamine N-acetyltransferase

Biochemical Journal ◽

10.1042/bj2970441 ◽

1994 ◽

Vol 297 (3) ◽

pp. 441-445 ◽

Cited By ~ 64

Author(s):

D Hickman ◽

A Risch ◽

V Buckle ◽

N K Spurr ◽

S J Jeremiah ◽

...

Keyword(s):

Bladder Cancer ◽

In Situ Hybridization ◽

Yeast Artificial Chromosome ◽

Chromosomal Localization ◽

Artificial Chromosome ◽

Acetylator Phenotype ◽

Chromosome 8 ◽

Slow Acetylator ◽

Human Genes

Arylamine N-acetyltransferase is encoded at two loci, AAC-1 and AAC-2, on human chromosome 8. The products of the two loci are able to catalyse N-acetylation of arylamine carcinogens, such as benzidine and other xenobiotics. AAC-2 is polymorphic and individuals carrying the slow-acetylator phenotype are more susceptible to benzidine-induced bladder cancer. We have identified yeast artificial chromosome clones encoding AAC-1 and AAC-2 and have used the cloned DNAs as fluorescent probes for in situ hybridization. The hybridization patterns allow assignment of AAC-1 and AAC-2 to chromosome 8p21.3-23.1, a region in which deletions have been associated with bladder cancer [Knowles, Shaw and Proctor (1993) Oncogene 8, 1357-1364].

Download Full-text

Compositional heterogeneity and patterns of molecular evolution in the Drosophila genome.

Genetics ◽

10.1093/genetics/134.3.837 ◽

1993 ◽

Vol 134 (3) ◽

pp. 837-845

Author(s):

J P Carulli ◽

D E Krane ◽

D L Hartl ◽

H Ochman

Keyword(s):

Molecular Evolution ◽

Base Composition ◽

Yeast Artificial Chromosome ◽

Strong Support ◽

Direct Determination ◽

Artificial Chromosome ◽

Significant Heterogeneity ◽

Sequence Evolution ◽

Drosophila Genome ◽

Eukaryotic Organisms

Abstract The rates and patterns of molecular evolution in many eukaryotic organisms have been shown to be influenced by the compartmentalization of their genomes into fractions of distinct base composition and mutational properties. We have examined the Drosophila genome to explore relationships between the nucleotide content of large chromosomal segments and the base composition and rate of evolution of genes within those segments. Direct determination of the G + C contents of yeast artificial chromosome clones containing inserts of Drosophila melanogaster DNA ranging from 140-340 kb revealed significant heterogeneity in base composition. The G + C content of the large segments studied ranged from 36.9% G + C for a clone containing the hunchback locus in polytene region 85, to 50.9% G + C for a clone that includes the rosy region in polytene region 87. Unlike other organisms, however, there was no significant correlation between the base composition of large chromosomal regions and the base composition at fourfold degenerate nucleotide sites of genes encompassed within those regions. Despite the situation seen in mammals, there was also no significant association between base composition and rate of nucleotide substitution. These results suggest that nucleotide sequence evolution in Drosophila differs from that of many vertebrates and does not reflect distinct mutational biases, as a function of base composition, in different genomic regions. Significant negative correlations between codon-usage bias and rates of synonymous site divergence, however, provide strong support for an argument that selection among alternative codons may be a major contributor to variability in evolutionary rates within Drosophila genomes.

Download Full-text