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PubMed2005

Phylogenetic information in polymorphic L1 and Alu insertions from East Asians and Native American populations.

This study attempts to ascertain genetic affinities between Native American and East Asian populations by analyzing four polymorphic Alu insertions (PAIs) and three L1 polymorphic loci. These two genetic systems demonstrated strong congruence when levels of diversity and genetic distances were considered. Overall, genetic relatedness within Native American groups does not correlate with geographical and linguistic structure, although strong grouping for Native Americans with East Asians was demonstrated, with clear discrimination from African and European groups. Most of the variation was assigned to differences occurring within groups, but the interpopulation variation found for South Amerindians was recognizably higher in comparison to the other sampled groups of populations. Our data suggest that bottleneck events followed by strong influence of genetic drift in the process of the peopling of the Americas may have been determinant factors in delineating the genetic background of present-day South Amerindians. Since no clear subgroups were detected within Native Americans and East Asians, there is no indication of multiple waves in the early colonization of the New World.

Alu Elements↗
PubMed2003

Two problematic human polymorphic Alu insertions.

Analysis of two previously described polymorphic Alu insertions (Sb19.3 and NBC3) in world-wide human populations generated genotypic frequencies grossly in violation of Hardy-Weinberg equilibrium expectations. GenBank searches at the National Center for Biotechnology Information (NCBI) and sequencing analyses revealed that samples homozygous for the Sb19.3 Alu insertion amplify a band indistinguishable in size to the lack of insertion amplicon, corresponding to a paralogous locus on chromosome 4. This locus displays a very similar sequence (84%) to that flanking the Sb19.3 Alu insertion located at chromosome 19. Moreover, we have determined that NBC3, a different Alu insertion, is not located in the pseudoautosomal region of the Y-chromosome, as previously reported, but in position Yq11.2. Also, the band that mimics the lack of insertion amplicon corresponds to a paralogous locus located at chromosome X with a similarity of 92% to the sequence flanking the NBC3 Alu insertion. Finally, the utilization of newly designed primers avoided amplification from the paralogous loci and allowed a reliable assignation of genotypes for both loci. Unlike previously reported, using our new primers the Y-specific locus NBC3 was found not to be polymorphic in the populations analyzed.

Alu Elements↗
PubMed2001

A recurrent large Alu-mediated deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene associated with Lesch-Nyhan syndrome.

We identified the identical large genomic deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene in two Japanese patients with Lesch-Nyhan (LN) syndrome. This deletion spanned from an Alu sequence in the promoter region to another Alu-sequence in intron 1, a length of 2,969 base pairs including exon 1. In order to ask whether this deletion was a recurrent mutation, we developed a simple alternative method to determine the separate origin of the HPRT1 mutation of the patients as assessed with an apparent mtDNA polymorphism. Considering that an LN syndrome-causing mutation is not transmitted from patient to offspring as LN syndrome is a fatal disease in childhood and that mtDNA is maternally inherited, HPRT1 mutations and mtDNA would be co-transmitted from carrier mother to offspring since both appeared in females. Two bases were different in the hypervariable region I of the mtDNA between the two patients, indicating the separate origin of their mtDNA over at least several thousand years as calculated based on the molecular evolution rate in this region. We thus conclude that the identical deletion found in HPRT1 of the two patients was derived from recurrent events of genomic recombination. Given that the same Alu-mediated deletion of HPRT1 has not been reported among somatic mutations at the same locus, this region of the HPRT1 gene flanked by Alu-sequences is likely a mutational hot spot in the germline but not in somatic cells. In addition, we also report novel LN-syndrome-conferring mutations in intron 6 (IVS6+1G --> C) and intron 8 (IVS7-9T --> G) that resulted in exclusions of exon 6 and exon 8, respectively.

Alternative Splicing↗
PubMed2005

The 5' region of the MSH2 gene involved in hereditary non-polyposis colorectal cancer contains a high density of recombinogenic sequences.

MSH2 rearrangements are involved in approximately 10% of hereditary non-polyposis colorectal cancer (HNPCC) families, and in most of the rearrangements, exon 1 is deleted. We scanned by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) 200 kb of genomic sequences upstream of the MSH2 transcription initiation site in 21 HNPCC families with exon 1 deletions. This QMPSF scan revealed 12 distinct 5' breakpoints located up to 200 kb upstream of the MSH2 transcription initiation site. Sequencing analysis of the rearranged allele in 17 families revealed that most of the deletions (15/17) resulted from homologous Alu-mediated recombination. QMPSF and sequencing analysis in these 21 families led us to detect the presence of 20 distinct 5' breakpoints. In 14 out of 15 Alu-mediated recombinations, we found, either within the identical region in which the recombination had probably occurred or in its vicinity, the 26-bp Alu core sequence containing the recombinogenic Chi-like motif. Compared to the equivalent regions of other human genes, the MSH2 upstream region was found to contain a high density of Alu repeats (30% within 228 kb and 43% within 50 kb), most of which belong to the old Alu S subfamilies. In conclusion, this study demonstrates the heterogeneity of the breakpoints within the MSH2 upstream region and reveals the remarkable density of recombinogenic Alu sequences in this region.

Adult↗
PubMed2003

Hereditary nonpolyposis colorectal cancer: frequent occurrence of large genomic deletions in MSH2 and MLH1 genes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease-causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu-repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Inter-alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatin.

Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub-lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter-alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter-alu PCR) can determine the nature of alterations being assessed. We have compared the inter-alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter-alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p = 1.866 x 10(-4)). The number of changes observed with respect to the corresponding leucocyte DNA in the inter-alu PCR profile of 26 primary tumors (Grade II = 13; Grade IV = 13), resected before chemotherapy, for the 2 inter-alu primers was very small. We present a novel application of the inter-alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub-lethal doses of cisplatin but not in the genesis of primary glioma.

Alu Elements↗
PubMed2006

Diversity of contactin mRNA in human brain tumors.

In order to address the molecular signature of human glioma, we investigated the polymorphism of 5'UTR of the mRNA of Contactin, an adhesion molecule which plays a role in the invasive behavior of these tumors. Contactin mRNA is identified by RT-PCR and a strategy based on rapid amplification of cDNA ends (RACE) reveals different 5'UTRs resulting from both an alternative use of two types of leader exons and a splicing mechanism within the 5'UTR. The spliced exon is an Alu-containing element specific to the primate lineage, thus indicating a recent evolution of regulatory processes specific to the simian line that occurs on this gene. Each 5'UTR exhibits different transcription/translation efficiencies and contains features that allow translation to occur independently of the classic cap-dependent mechanism. These data illustrate the complex regulation of Contactin expression in human brain tumors occurring at both transcriptional and translation levels. The different 5'UTRs are differentially expressed in diverse types of human tumors. Thus, the polymorphism occurring within the non-coding part of the Contactin mRNA reveals new opportunities to explore deregulation that occurs during the oncogenic process.

5' Untranslated Regions↗
PubMed1995

The baboon expresses the calbindin-D9k gene in intestine but not in uterus and placenta: implication for conservation of the gene in primates.

Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5' end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues.

Animals↗
PubMed1999

Genomic organization of human myristoyl-CoA: protein N-myristoyltransferase-1.

Myristoylation is a biochemical modification of proteins in which the lipid myristate becomes covalently bound to various cellular, viral, and oncoproteins catalyzed by a monomeric enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). This modification is important for the biological activity of several proteins, especially the regulation of several oncoproteins involved in various types of cancers. Complementary DNA encoding human NMT-1 (hNMT-1) has been previously reported; however, the genomic organization of hNMT-1 has not been available. Attempts to amplify genomic fragments corresponding to hNMT-1 cDNA sequence yielded only one fragment. We have searched databases using both the cDNA and sequence of one of the intron sequence and this identified a human BAC clone sequence from chromosome 17. Alignment of hNMT-1 cDNA coding information on human chromosome 17 resulted in the complete structural identity of 23,960 bp of the hNMT-1 gene. The hNMT-1 gene is composed of 11 exons and 10 introns with consensus GT/AG boundaries. Finally, we show that 140 bp from the 5' end of recently reported full-length cDNA of hNMT-1 was not part of this genomic region raising the possibility for posttranscriptional modification in generating larger transcripts likely by trans splicing. Further, the availability of this genomic sequence will assist in unraveling the molecular basis for several observed NMT isoforms.

Acyl Coenzyme A↗
PubMed1999

Sequence analysis of the spacer region between the RHD and RHCE genes.

Numerous variants of the Rh blood group system, discovered by Levine and Stetson in 1939, have been detected and more than forty antigens have been identified. By performing the molecular genetic analysis of the introns as well as the exons in both RH genes, it was elucidated that Rh variants were generated by gene conversion or recombination, deletions, or mutations. For understanding the generation of many Rh variants and Rh antigens in detail, it is necessary to analyze not only the RHCE and RHD genes but also the structure and the physical distance between both these RH genes. In order to achieve the aforesaid purpose, the spacer region between the RHD and RHCE genes were amplified by the long PCR method. Therefore the full spacer region was determined to be 12159 bp in length and contained the Alu consensus sequences and the putative CpG island. It was probable that the duplication of both RH genes occurred within about 12 kb region. Analysis of the spacer region provides new information for the research on the transcription-control region, the molecular evolution of RH genes, Rh variants, and the deletion of the RHD gene in Rh blood group system.

Alu Elements↗
PubMed1999

Identification and characterization of a transcriptional silencer upstream of the human BRCA2 gene.

Expression of the breast cancer susceptibility tumor-suppressor protein BRCA2, a protein potentially involved in DNA recombination repair, is tightly regulated throughout development. We have identified a transcriptional silencer at the distal end of the human BRCA2 gene promoter. This silencer is involved in the negative regulation of the expression of this gene in breast cell lines tested but not in HeLa or HepG2 cells. The 221-base-pair silencer region is characterized by a full-length Alu-repeat. Presence of specific BRCA2 silencer-binding proteins in the breast cell extracts indicates the potential regulation of BRCA2 gene expression by these proteins.

Alu Elements↗
PubMed1996

The entire genomic sequence and cDNA expression of mouse alpha-galactosidase A.

The full-length cDNA and genomic sequences encoding mouse alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22), a lysosomal galactohydrolase, were isolated and characterized. The cDNA's open reading frame encoded 419 amino acids and had 82% nucleotide (nt) and 78% amino acid identity with the human sequence, although the carboxy terminus of the mouse alpha-Gal A polypeptide was 10 amino acids shorter. The functional integrity of the mouse cDNA was demonstrated by transient expression in COS-1 cells. Northern analysis revealed two mRNA species of about 1.6 and 3.4 kb due to alternative polyadenylation signals. The entire 14.4-kb mouse genomic sequence was determined; each of its seven exons was interrupted by intronic sequence at the identical positions as the exons in the human gene. The mouse 5' flanking region (250 nt) had one Sp1, site, five CAAT boxes, and no TATA box and had 67% identity with the human promoter region. The gene contained 18 complete or partial Alu-repetitive elements (13 type 1 and 5 type 2 repeats), and three putative functional AATAAA consensus polyadenylation signals were identified 72, 1668, and 1682 nt after the TAA termination codon. Use of the 72-nt site and the 1866 and/or 1682 sites were consistent with the shorter and longer transcripts. The availability of the full-length cDNA and genomic sequence encoding mouse alpha-Gal A should facilitate structure/function studies of this lysosomal glycosidase and the construction of alpha-Gal A-deficient mice by targeted gene disruption.

Amino Acid Sequence↗
PubMed1993

Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene.

Acute intermittent porphyria (AIP), an autosomal dominant inborn error of heme biosynthesis, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (HMB-synthase). Heterozygous individuals are prone to life-threatening acute neurologic attacks, which are precipitated by certain drugs and other metabolic, hormonal, and nutritional factors. Since the biochemical diagnosis of heterozygous individuals has been problematic, recent efforts have focused on the identification of mutations and diagnostically useful restriction fragment length polymorphisms (RFLPs) in the HMB-synthase gene. To facilitate these endeavors, the human HMB-synthase gene, including 1.1 kg of the 5' flanking region, was isolated and completely sequenced in both orientations. The 10,024-bp gene contained 15 exons ranging in size from 39 to 438 bp and 14 introns ranging from 87 to 2913 bp. All intron/exon boundaries conformed to the GT/AG consensus rule. There were six Alu repetitive elements, one of the J and five of the Sa subfamilies. Analysis of the 1.1-kb 5' flanking region revealed putative regulatory elements for the housekeeping promoter including AP1, AP4, SP1, TRE, ENH, and CAC. This region contained 10 HpaII sites and had an overall GC content of 54%. Intron 1, which contained the erythroid-specific promoter, had putative regulatory motifs for NF-1, NF-E1, NF-E1(b), NF-E2, AP1, AP4, TOPO, CAAC, CAC, CAAT, and TATA. The locations and variant nucleotides for the known RFLPs in intron 1 were identified [MspI, nucleotide 1345 G/A; PstI, 1500 C/T; ApaLI, 2377 C/A; and BstNI, 2479 G/A] and improved polymerase chain reaction (PCR)-based detection methods for each were established.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1994

Human delta-aminolevulinate dehydratase (ALAD) gene: structure and alternative splicing of the erythroid and housekeeping mRNAs.

Genomic clones containing human delta-aminolevulinate dehydratase (ALAD), the second enzyme in the heme pathway, were isolated, and the entire sequence was determined in both orientations (15,913 bp; GenBank Accession No. X64467). The gene contained two alternative noncoding exons, 1A and 1B, and 11 coding exons, 2-12. Ten Alu-repetitive elements were within the gene, including an inverted repeat that may have resulted from gene conversion. The housekeeping transcript, which included exon 1A and not 1B, was identified in a human adult liver cDNA library, while an erythroid-specific transcript, which contained exon 1B and not 1A, was detected in a human K562 erythroleukemia cDNA library. The promoter region upstream of housekeeping exon 1A was GC-rich and contained three potential Sp1 elements and a CCAAT box. Further upstream, there were three potential GATA-1 binding sites and an AP1 site. The promoter region upstream of erythroid-specific exon 1B had several CACCC boxes and two potential GATA-1 binding sites. To assess the tissue-specific expression of exons 1A and 1B, HeLa and K562 cells were transduced with CAT constructs containing either exon 1A or 1B and their respective upstream promoter region. Two housekeeping CAT constructs, with 450 and 1400 bp upstream of exon 1A, were expressed at similar levels in HeLa cells, whereas the erythroid-specific construct, containing the entire 450-bp promoter region upstream of exon 1B, was not. In contrast, the housekeeping and erythroid constructs were both expressed in K562 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Development of 124 sequence-tagged sites and cytogenetic localization of 217 cosmids for human chromosome 10.

A total of 124 new chromosome 10-specific sequence-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched lambda phage library (LL10NS01) and the second by cloning Alu element-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished from Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or of YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats.

Base Sequence↗
PubMed1994

The structure and organization of the human erythroid anion exchanger (AE1) gene.

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans approximately 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3' noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.

Amino Acid Sequence↗
PubMed1996

Analysis of three deletion breakpoints in Xp21.1 and the further localization of RP3.

The gene responsible for X-linked retinitis pigmentosa (xlRP) in Xp21.1 (RP3) was initially localized by deletion analysis to within a 150- to 170-kb region between the CYBB locus and the proximal deletion junction (BBJPROX) from a patient, BB, who suffered from Duchenne muscular dystrophy (DMD), McLeod syndrome, chronic granulomatous disease (CGD), and xlRP. This gene has recently been isolated and was found to be located outside and 400 kb proximal to the BB deletion. Further analysis of BBJPROX has identified the breakpoint junction sequence, showing that it occurs within an Alu repetitive element on the proximal side but with no significant homology to the distal sequence in dystrophin intron 30. Analysis of an overlapping deletion in patient NF, who suffered from DMD, CGD, and McLeod syndrome, shows that this deletion is within 4 kb but extends centromeric to BBJPROX, consistent with the location of RP3 outside the BB deletion region. A sequence with strong homology to a THE-1 transposon-like element was identified 7-13 kb from the proximal BB and NF breakpoints. These elements have been implicated in several highly unstable genomic regions. A third overlapping deletion, in a patient, SB, who suffered from CGD, McLeod syndrome, and xlRP, has here been shown to extend 380 kb proximal to the NF breakpoint, consistent with the finding that RP3 lies outside the BB deletion. This deletion has now been shown to disrupt the RP3 (RPGR) gene. The reason for the retinitis pigmentosa phenotype in patient BB remains unclear, but the most likely explanations include a long-range chromosomal position effect, a small secondary rearrangement, and the presence of a coincident autosomal form of retinitis pigmentosa.

Base Sequence↗