The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.
The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.
Blackshear
P J
PJ
Office of Clinical Research and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, 111 Alexander Drive, Research Triangle Park, NC 27709, USA. black009@niehs.nih.gov
Lai
W S
WS
Thorn
J M
JM
Kennington
E A
EA
Staffa
N G
NG
Moore
D T
DT
Bouffard
G G
GG
Beckstrom-Sternberg
S M
SM
Touchman
J W
JW
Bonaldo
M F
MF
Soares
M B
MB
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D016208:Databases, Factual; D004782:Environmental Health; D020224:Expressed Sequence Tags; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005787:Gene Frequency; D015723:Gene Library; D014644:Genetic Variation; D008969:Molecular Sequence Data; D009316:National Institutes of Health (U.S.); D010063:Ovum / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014481:United States; D014981:Xenopus / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger