ABSTRACT
A YAC/STS map of the X chromosome has reached an inter-STS resolution of 75 kb. The map density is sufficient to provide YACs or other large-insert clones that are cross-validated as sequencing substrates across the chromosome. Marker density also permits estimates of regional gene content and a detailed comparison of genetic and physical map distances. Five regions are detected with relatively high G + C, correlated with gene richness; and a 17-Mb region with very low recombination is revealed between the Xq13.3 [XIST] and Xq21.3 XY homology loci.
Subject(s)
Chromosome Mapping , X Chromosome/genetics , Base Composition/genetics , Chromosome Mapping/methods , Chromosomes, Artificial, Yeast/genetics , Cytosine Nucleotides/genetics , DNA, Complementary/genetics , Gene Expression/genetics , Genomic Library , Guanine Nucleotides/genetics , Humans , Sequence Tagged SitesABSTRACT
DNA comprising 219 447 bp was sequenced in nine cosmids and verified at > 99.9% precision. Of the standard repetitive elements, 187 Alus make up 20.6% of the sequence, but there were only 27 MERs (2.9%) and 17 L1 fragments (1.6%). This may be characteristic of such high GC (57%) regions. The sequence also includes an 11.3 kb tract duplicated with 99.2% identity at a distance of 38 kb. The region is 80-90% transcribed and 12.5% translated. Thirteen known genes and their exon-intron borders are all accurately predicted at least in part by GRAIL programs, as are six additional genes. From centromere to telomere, the orientation of transcription varies among the first eight genes, then runs centromeric to telomeric for the next five, and is in the opposite sense for the last six. Eighteen of the 19 genes are associated with CpG islands. Two islands are exact copies in the 11.3 kb repeat units, and could thus give rise to double dosage levels of an X-linked gene. Another island is associated with two genes transcribed in opposite directions. From the sequence data, three genes and their exon structure are inferred. One of them, previously associated with HEX2, is shown to be a different gene unrelated to hexokinases; a second gene, previously known by an EST, is plexin, from its 65.5% identity with the Xenopus analog; and a third is a subunit of a vacuolar H-ATPase, and is named VATPS1.
Subject(s)
Color Perception/genetics , Cytidine , Glucosephosphate Dehydrogenase/genetics , Guanosine , X Chromosome , Base Sequence , Cosmids/chemistry , CpG Islands , DNA , Dinucleotide Repeats , Female , Humans , Introns , Molecular Sequence Data , SoftwareABSTRACT
Screening of collections of yeast artificial chromosomes utilizing the polymerase chain reaction (PCR) requires large numbers of reactions in parallel. Four steps were implemented to reduce the labor involved: (a) The number of initial samples for DNA extractions was decreased by compressing libraries up to 12-fold. (b) DNA extraction from yeast clones was robot assisted. (c) A BIOMEK 1000 station was adapted to pipette samples for PCR assays. (d) Sample preparation was integrated with a temperature cycler constructed to carry out up to 576 reactions in six 8 x 12-well trays. The implementation of these steps increases the number of reactions per person per day by an order of magnitude. In tests with X-chromosome-specific probes, the robot-aided screening recovered all of the clones detected by slower manual methods.
