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Results for “Alu elements”

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PubMed1989

The human lysozyme gene. Sequence organization and chromosomal localization.

We have isolated two overlapping recombinant lambda-phage clones from a genomic lambda-EMBL3 library containing 25 kb of the human lysozyme gene region. Furthermore a full-lenght human lysozyme cDNA clone of 1.5 kb was isolated from a human placenta cDNA library. Nucleotide sequences of the entire structural gene and the cDNA clone were determined. The human lysozyme gene spans 5856 bp and its sequence organization with four exons and three introns is homologous to the chicken lysozyme gene and the human alpha-lactalbumin gene. Human and chicken lysozyme genes differ mainly in the size of their introns and 3' non-coding region. Four Alu repetitive elements were found in the human lysozyme gene, one in each intron and one on the fourth exon. Lysozyme transcripts of 1.6 kb and 0.6 kb in size were detected in human myeloid cell lines U-937, HL-60 and THP-1 and surprisingly in human hepatoma cell lines HepG2 and Hep3B. The lysozyme gene locus was assigned to human chromosome 12 by hybridization to a panel of DNAs from human-rodent somatic cell hybrids.

Animals↗
PubMed1997

Structure and expression of the gene encoding secretor-type galactoside 2-alpha-L-fucosyltransferase (FUT2).

The expression and secretion of ABO antigens in epithelial cells of glands are controlled by secretor-type alpha (1,2)fucosyltransferase activity. We have examined the expression of the secretor-type alpha(1,2)fucosyltransferase gene (FUT2) and a pseudogene of FUT2 (Sec1) in several tumor cell lines by northern blot and/or reverse-transcription-PCR (RT-PCR) analyses. Transcripts of FUT2 were found in total RNA from ovarian, gastric and colonic cancer cell lines but not from six leukemic cell lines, including erythroleukemic HEL cells, by RT-PCR. On the other hand, RT-PCR indicated that Sec1 was expressed in all these tumor cells, including all hematopoietic cells studied. Northern blot analysis indicated that FUT2 transcripts with a similar size (3.3 kb) were expressed in cancer cell lines. Rapid amplification of cDNA ends suggested that the entire FUT2 cDNA is 3.1-kb long and has two Alu repetitive elements in its 3' untranslated region, including an inverted repeat. The mRNA, therefore, may form a large stem-and-loop structure (1.2 kb). Each stem contains about 300 bases, the loop contains 640 bases, and the percentage of complementary nucleotide sequences in the stem region is 85%. The presence of a large stem-and-loop structure in the 3' untranslated region may regulate the level of the FUT2 transcript by affecting the stability of the mRNA.

Animals↗
PubMed2006

Genetic change in the polynesian population of Easter Island: evidence from Alu insertion polymorphisms.

The origin of Pacific islanders is still an open issue in human population genetics. To address this topic we analyzed a set of 18 Alu insertion polymorphisms in a total of 176 chromosomes from native Easter Island inhabitants (Rapanui). Available genealogical records allowed us to subdivide the total island sample into two groups, representative of the native population living in the island around 1900, and another formed by individuals with some ancestors of non-Rapanui origin. Significant genetic differentiation was found between these groups, allowing us to make some biodemographic and historical inferences about the origin and evolution of this geographically isolated island population. Our data are consistent with equivalent and recent contributions from Amerindian and European migrants to the 1900s Rapanui population, with an accelerated increase in the European gene flow during the 20(th) century, especially since the 1960s. Comparative analysis of our results with other available Alu variation data on neighbouring populations supports the "Voyaging Corridor" model of Polynesian human settlement, which indicates that pre-Polynesians are mainly derived from Southeast Asian and Wallacean populations rather than from Taiwan or the Philippines. This study underlines the importance of sampling and taking into account historical information in genetic studies to unravel the recent evolution of human populations.

Alu Elements↗
PubMed2006

Simultaneous determination of total human and male DNA using a duplex real-time PCR assay.

A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.

Alu Elements↗
PubMed2000

Genetics. L1 retrotransposons shape the mammalian genome.

What are all of those retrotransposons doing buzzing about in our genome? According to Kazazian, in his Perspective this week, these mobile pieces of DNA are busy reshaping our genome, making it more diverse and enabling us to survive and thrive through the vagaries of evolution. And just how do they do this?...zip to page 1152 to find out.

Alu Elements↗
PubMed1985

Reverse transcription of 7S L RNA by an avian retrovirus.

Linial et al. isolated a quail cell line, SE21Q1b, that is transformed by a single integrated provirus of Rous sarcoma virus. Virus particles are released from these cells, but because of a provirus defect, cellular rather than viral RNA is packaged. When these virus particles are disrupted with melittin in the presence of an appropriate reaction mixture containing actinomycin D, there is significant reverse transcription of packaged cellular RNA species. We have shown that (i) cellular 7S L RNA is an efficient template; (ii) initiation is on a unique tRNA-like primer; (iii) synthesis produces a 135-base strong-stop DNA product; and (iv) after synthesis, RNase H acts to remove the 135 bases of the 7S L RNA which acted as the template. A possible facilitator of such specific transcription may be that, in the virus particles but not in the cell, the majority of the 7S L RNA species exist complexed with the tRNA, even before the disruption of the virus. From the size and sequence features of the reverse transcript of 7S L RNA, we speculate that such events may have participated in the process by which animal cell genomes have, in the course of evolution, accumulated multiple copies of Alu-like elements.

Avian Sarcoma Viruses↗
PubMed2001

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase.

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.

3T3 Cells↗
PubMed2001

The signal recognition particle.

The signal recognition particle (SRP) and its membrane-associated receptor (SR) catalyze targeting of nascent secretory and membrane proteins to the protein translocation apparatus of the cell. Components of the SRP pathway and salient features of the molecular mechanism of SRP-dependent protein targeting are conserved in all three kingdoms of life. Recent advances in the structure determination of a number of key components in the eukaryotic and prokaryotic SRP pathway provide new insight into the molecular basis of SRP function, and they set the stage for future work toward an integrated picture that takes into account the dynamic and contextual properties of this remarkable cellular machine.

Alu Elements↗
PubMed2002

Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2.

Casein kinase 2 (CK2) is a tetrameric enzyme constitutively expressed in all eukaryotic tissues. The two known isoforms of the catalytic subunit, CK2alpha and CK2alpha', have been reported to have distinct tissue-dependent subcellular distributions. We recently described a third isoform of the catalytic subunit, designated CK2alpha", which is highly expressed in liver. Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as immunofluorescent microscopy revealed that CK2alpha" was exclusively localized to the nucleus and preferentially associated with the nuclear matrix. CK2alpha and CK2alpha' were found in nuclear, membrane, and cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2alpha" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclear retention indicates that the underlying mechanism of CK2alpha" nuclear localization is dependent on the secondary structure of the holoenzyme directed by the carboxy-terminal sequence.

Active Transport, Cell Nucleus↗
PubMed1997

Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization.

Many purine nucleosides and their analogs are actively transported in the kidney. Using homology cloning strategies and reverse transcriptase-polymerase chain reactions, we isolated a cDNA encoding a Na(+)-dependent nucleoside transporter, hSPNT1, from human kidney. Functional expression in Xenopus laevis oocytes identified hSPNT1 as a Na(+)-dependent nucleoside transporter that selectively transports purine nucleosides but also transports uridine. The Michaelis constant (K(m)) of uridine (80 microM) in interacting with hSPNT1 was substantially higher than that of inosine (4.5 microM). hSPNT1 (658 amino acids) is 81% identical to the previously cloned rat Na(+)-nucleoside transporter, SPNT, but differs markedly from SPNT in terms of its primary structure in the NH2 terminus. In addition, an Alu repetitive element (approximately 282 bp) is present in the 3'-untranslated region of the hSPNT1 cDNA. Northern analysis revealed that multiple transcripts of hSPNT1 are widely distributed in human tissues including human kidney. In contrast, rat SPNT transcripts are absent in kidney and highly localized to liver and intestine. The hSPNT1 gene was localized to chromosome 15. This is the first demonstration of a purine nucleoside transporter in human kidney.

Adenosine↗
PubMed2004

A comparison of oligonucleotide and cDNA-based microarray systems.

Large-scale public data mining will become more common as public release of microarray data sets becomes a corequisite for publication. Therefore, there is an urgent need to clarify whether data from different microarray platforms are comparable. To assess the compatibility of microarray data, results were compared from the two main types of high-throughput microarray expression technologies, namely, an oligonucleotide-based and a cDNA-based platform, using RNA obtained from complex tissue (human colonic mucosa) of five individuals. From 715 sequence-verified genes represented on both platforms, 64% of the genes matched in "present" or "absent" calls made by both platforms. Calls were influenced by spurious signals caused by Alu repeats in cDNA clones, clone annotation errors, or matched probes that were designed to different regions of the gene; however, these factors could not completely account for the level of call discordance observed. Expression levels in sequence-verified, platform-overlapping genes were not related, as demonstrated by weakly positive rank order correlation. This study demonstrates that there is only moderate overlap in the results from the two array systems. This fact should be carefully considered when performing large-scale analyses on data originating from different microarray platforms.

Aged↗
PubMed2006

A sensitive method to quantify human long DNA in stool: relevance to colorectal cancer screening.

Human long DNA in stool may reflect nonapoptotic exfoliation and has been used as a colorectal cancer (CRC) marker. Targeting human-specific Alu repeats represents a logical but untested approach. A real-time Alu PCR assay was developed for quantifying long human DNA in stool and evaluated in this study. The accuracy and reproducibility of this assay and the stability of long DNA during room temperature fecal storage were assessed using selected patient stools and stools added to human DNA. Thereafter, long DNA levels were determined in blinded fashion from 18 CRC patients and 20 colonoscopically normal controls. Reproducibility of real-time Alu PCR for quantifying fecal long DNA was high (r2 = 0.99; P < 0.01). Long DNA levels in nonbuffered stools stored at room temperature fell a median of 75% by 1 day and 81% by 3 days. An EDTA buffer preserved DNA integrity during such storage. Human long DNA was quantifiable in all stools but was significantly higher in stools from CRC patients than from normal controls (P < 0.05). At a specificity of 100%, the sensitivity of long DNA for CRC was 44%. Results indicate that real-time Alu PCR is a simple method to sensitively quantify long human DNA in stool. This study shows that not all CRCs are associated with increased fecal levels of long DNA. Long DNA degrades with fecal storage, and measures to stabilize this analyte must be considered for optimal use of this marker.

Alu Elements↗
PubMed2006

Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60 degrees C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process.

Alu Elements↗