A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)+ RNAs but not for poly(A)− RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.
Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor.
Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was ∼ 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.
The C-terminal half molecule (C lobe) of bovine lactoferrin was isolated by mild tryptic hydrolysis of lactoferrin followed by gel filtration and ion-exchange chromatography. The identity of the fragment was established by determining its N-terminal and C-terminal amino acid sequences and comparing them with the amino acid sequence of intact lactoferrin. The isoelectric point of the C lobe ranged between pH 6.2 and 6.5 as measured by isoelectric focusing on polyacrylamide gels. The circular dichroic spectrum in the range of 250 to 350 nm of the C lobe differed slightly from that of intact lactoferrin. The pattern of lectin reactivity was similar for both the C lobe and intact lactoferrin. The C lobe showed partial antigenic identity with intact lactoferrin as demonstrated by the double immunodiffusion method, and pH dependence of iron binding of C lobe is the same as that of intact lactoferrin molecule.
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