A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen
Mara HareuveniIlan TsarfatyJoseph Z. ZaretskyP KotkesJudith HorevS. ZrihanMordechai WeissStephen GreenRichard LatheIafa KeydarDaniel H. Wreschner
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A monoclonal antibody, H23, that specifically recognizes a breast‐tumor‐associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850‐bp cDNA insert, shows that they are composed of 60‐bp (G + C)‐rich tandem repeating units. The coding strand was determined and codes for a proline‐rich 20‐amino‐acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60‐bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60‐bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragement produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over‐expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.Keywords:
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Two allelic p53 genes were investigated. The allelic gene with BgIII site in the first intron codes for faster p53 protein variant, the other allelic gene coding for slower p53 protein variant (according to the mobility in SDS-PAAG). Exons and flanked regions of introns were sequenced. In coding regions allelic genes only differ by second nucleotides of codon 72 (G----C), which leads to the change in amino acid sequence (Arg----Pro). The relationship of these changes and the function of p53 is discussed.
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Abstract We constructed a “cDNA bank” of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients’ tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 °C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.
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To investigate the features and functions of Sox 9 genes which are related to sex-determination and bony-tissue-development in C.carpio,several CcSox 9 genes were cloned from large quantity of individuals of several usual C.carpio varieties and wild Yellow River C.carpio.Sequences,including promoter-sequences,polymorphism,of these CcSox 9 genes and other CcSox 9 genes cloned by other domestic and foreign areas were analyzed.The results revealed that at least 5 active CcSox 9 genes and a pseudo gene PCcSox 9 were found.Abundant polymorphisms,including sequence polymorphisms and length polymorphisms,were found in these genes' coding sequences,especially introns.At least one promoter sequence can be found in each active CcSox 9 gene's intron.Pseudo gene PCcSox 9 has no intron and several terminal codons were found in its coding sequence.
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The c-H-ras-1 gene of an B6C3F1 mouse was isolated and nucleotide sequence determined. Our study has revealed that this c-H-ras-1 gene consists of four exons, separated by three introns ranging in size from 150 to 649 bp. The coding parts of the sequence of mouse c-H-ras-1 gene show no important differences as compared with those of the rat, hamster and human gene. More numerous changes were found in introns. The identity of mouse c-H-ras-1 gene with rat, hamster and human ones at the nucleotide level is 86.40%, 80.04% and 67.87%, respectively. Comparison of amino acids in protein sequence of c-H-ras gene of mouse, rat, hamster and human points to high degree of conservation of the gene.
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A 3 kb cDNA coding for rat liver S ‐adenosylmethionine (AdoMet) synthetase has been isolated. The M r of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl‐Sepharose column. The length of the mRNA 5′ non‐coding region has been defined by primer‐extension analysis. The rat liver cloned cDNA has been also used to detect S ‐adenosylmethionine synthetase mRNA in human liver.
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