Cloning of Japanese quail ovalbumin cDNA in E. coli.
Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.
Klaudiny
J
J
Laboratory for Food Biotechnology Research, Slovak Academy of Sciences, Bratislava.
Klaudinyová
V
V
Hanes
J
J
Simúth
J
J
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D003370:Coturnix; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger; D010047:Ovalbumin; D004247:DNA