Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus.
Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.
Gilmore
R C
RC
Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.
Baker
J
J
Dempsey
S
S
Marchan
R
R
Corprew
R N
RN
Byrd
G
G
Maeda
N
N
Smithies
O
O
Bukoski
R D
RD
Harewood
K R
KR
Chatterjee
P K
PK
D000483:Alleles; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics; D020451:Contig Mapping; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D005787:Gene Frequency; D015723:Gene Library; D006162:Guanylate Cyclase / Q000235:genetics*; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012150:Polymorphism, Restriction Fragment Length; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D004251:DNA Transposable Elements; D004247:DNA; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C