Pseudogene

About: Pseudogene is a research topic. Over the lifetime, 5528 publications have been published within this topic receiving 336634 citations. The topic is also known as: Ψ & pseudogenes.

Rapid assessment of single-copy nuclear DNA variation in diverse species.

Abstract: We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin, MHC DQA, and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA), and in birds, reptiles and mammals (aldolase, H2AF, myoglobin). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A) or high (e.g. skink ALD-1) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for ‘targeted’ digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.

Identification of two human beta-tubulin isotypes.

Abstract: The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs.

Molecular cloning and characterization of human caspase-activated dnase

Abstract: Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.

Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells

Abstract: In many recent publications, it has been claimed that reverse transcriptase-polymerase chain reaction (RT-PCR) assays involving genes with tissue-restricted expression can be used for specific and sensitive detection of cancer cells in blood, bone marrow and lymph nodes. Many different target mRNAs have been evaluated for such purposes. One of the most extensively studied genes, CK19, is predominantly expressed in cells of epithelial origin and normally not at detectable levels in hematopoietic or lymphatic tissues. Based on previous reports on CK19 we wanted to establish a useful assay for detection of micrometastatic cells. RNA and DNA specimens extracted from peripheral blood nucleated cells of healthy volunteers, as well as cell lines positive and negative for CK19 expression, were used in nested RT-PCR assays. Using previously published primers, we found a novel pseudogene that shows a high degree of identity with the CK19 gene sequence, except for differences caused by 3 small deletions and a number of point mutations, resulting in termination codons and frameshifts. The gene has therefore no coding potential. Importantly, published primer sequences and reaction conditions used by several other groups to detect CK19 mRNA may have led to the amplification of this pseudogene. The data illustrate one of the problems that must be addressed in validating RT-PCR assays for micrometastasis detection, and it is suggested that previous work using CK19 as a marker should be reassessed in view of the present finding.