Expression of intermediate filament proteins during development of Xenopus laevis III. Identification of mRNAs encoding cytokeratins typical of complex epithelia
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Abstract A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55454; Mr ∼ 58 000 on SDS–PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3′-untranslated portion of the 2·4kb mRNA encoding this cytokeratin, designated XenCK55(5development of n blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCKl(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the ‘XK81 subfamily’ previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3′-untranslated region in Northern blots, SI nuclease and hybrid-selection-translation assays and found the ∼ 1·6kb XK81 mRNA and the resulting protein of Mr∼ 48 000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early development. However, their synthesis is not restricted to developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.Keywords:
Northern blot
Changes of expression in cold-regulated mRNA levels in potato (Solanum tuberosum L. cv. Superior) were analyzed. A total of 12,000 cDNAs was subjected to reverse Northern blot analysis using ^(32)P-dCTP- labeled first strand cDNA generated from the total RNA isolated from 25℃- and 4℃-treated potato plants. A total of 245 cDNA clones were sequenced from a cDNA library constructed from the cold treatment. Based on the BLAST results, 103 cDNA clones were found to be redundant. The analyzed genes were classified into 12 groups according to their putative functions, where the 20.2 % of group I was associated with energy metabolism, 13.1％ of group XI with cell rescue and defense, 2.6％ of group Ⅷwith signal transduction. Among the cDNA clones up-regulated by cold treatment from the results of the reverse Northern blot analysis, 32 were used for the Northern blot analysis. Most of the cold-treated clones showed overexpression compared with the control, while some showed down-regulation. In general, it was found that cold stress related genes were overexpressed more than two-folds at 4℃ treatment.
Northern blot
Solanum tuberosum
Southern blot
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African clawed frog
Model Organism
Complement
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Abstract We investigated the limitations and effectiveness of differential hybridization in the cloning of T cell‐specific cDNA (complementary DNA) molecular clones. By using the technique with T cell and B cell cDNA probes, together with Northern blot analysis, we successfully isolated cDNA clones exclusively expressed in T cells from 1 × 10 4 plaque‐forming units of a T cell hybridoma. These clones represent 0.068% of the mass of the cytoplasmic mRNA. Our result shows that differential hybridization is an effective procedure when used in combination with Northern blot analysis for screening of genes selectively expressed in T cells.
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Southern blot
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Differential display
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In this study using the technology of rapid amplification of cDNA ends(RACE),3′RACE and 5′RACE were amplified on the basis of the known partial reindeer ghrelin cDNA sequence from our team.The results indicated that the sequences of 3′and 5′cDNA end were amplified successfully,and obtained the 604 bp full-length sequence of reindeer ghrelin cDNA,including a 5′untranslated region(UTR)of 57 bp,a open reading frame(ORF)of 405 bp,3′UTR of 128 bp and ploy(A)14.The ORF of 405 bp encodes a preproghrelin of 134 amino acids.The predicted preproghrelin was composed of a N-terminal signal peptide of 41 amino acid residues,a 27 residues mature ghrelin peptide and a 66 residues C-terminal peptide.The sequence alignment showed that the cDNA of reindeer ghrelin has homology of 92.0%,90.8% and 89.5% with goat,sheep and cattle respectively,69.5%and 65.2% with pig and human,only 33.8% with chicken.The results indicate that the structure of ghrelin has a obvious varietal speciality.The obtaining of full-length reindeer ghrelin lay the foundation of further studying the function of physiology.
Cloning (programming)
Homology
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Southern blot
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An mRNA with a substantial similarity to the rat p62 mRNA that encodes a nucleoporin was cloned from the rat testis. A probe derived from a unique sequence in the nucleoporin-related (NPR) cDNA revealed a novel mRNA of 1.3 kb, different from the 2.7-kb transcript attributed to the p62 gene. This 1.3-kb transcript was not detected in Sertoli cells; it was found primarily in the haploid germ cells of the adult testis. The DNA sequencing revealed that the central region of the NPR cDNA sequence was identical to the 3' portion of the p62 cDNA containing heptad repeat sequences. However, the 5' region and the extreme 3' region of the NPR cDNA sequence were different from the p62 cDNA. Interestingly, the extreme 3' untranslated region (UTR) contained a 212-bp inverted repeat of a sequence located in the middle of the NPR cDNA that is identical to the p62 sequence. The inverted repeats of the NPR sequence could potentially hybridize, leading to the formation of circular transcripts. Using antibodies specific for the C-terminal regions of p62, a 26-kDa protein was detected from NPR cDNA hybrid-arrested translational products, and a 28-kDa protein was detected from the testis germ cell extracts but not from Sertoli cell extracts.
Nucleoporin
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A new cDNA clone for rat IGF-I mRNA was obtained. The cDNA contained a new base sequence as cDNA in the 3' region. The sequence coincided with a part of the sequence reported earlier by Shimatsu and Rotwein in exon 5 of the rat IGF-I gene. The presence of this cDNA proved the previous suggestion to be true that the size heterogeneity of IGF-I mRNA is primarily due to the heterogeneity of the 3'-untranslated region.
clone (Java method)
Sequence (biology)
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