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Characterization of the mouse matrilin-4 gene: a 5' antiparallel overlap with the gene encoding the transcription factor RBP-l.

We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5' UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5' with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.

5' Untranslated Regions↗

In vivo and in vitro expression of connexins in the human corneal epithelium.

PURPOSE: This study is designed to provide a comprehensive expression profile of connexins in the human corneal epithelium (in vivo) and in cultured primary corneal epithelial cells (PCECs) (in vitro). It also evaluates the pathologic effects of a pathogenic missense mutation in Cx26, which causes keratitis-ichthyosis-deafness syndrome (KIDS), a rare genetic disorder with corneal involvement. METHODS: RT-PCR analysis, immunohistochemistry, and fluorescent dye transfer assays were used to determine the expression pattern and gap junction intercellular communication in PCECs and human cornea. Differentiation-dependent differences in connexin expression of PCECs after a calcium switch were verified by real-time RT-PCR. The common KIDS mutation Cx26(D50N) was studied by determining transient expression in PCECs. RESULTS: In vivo immunostaining revealed widespread and overlapping expression of Cx43 and -30 in the basal and suprabasal layers. Cx26 staining was limited to the lower suprabasal cells, whereas Cx31.1 localized to the apical surface of basal cells in the central cornea and to the lower and middle suprabasal cells in the limbal region. Immunostaining for nine other connexins, including Cx50, was negative. In PCEC, nine connexin genes were detectable by RT-PCR, however, only Cx26, -30, and -43 formed visible gap junction plaques. High-Ca(2+) culture conditions were accompanied by a 1.6- to 2.2-fold upregulation of expression of Cx26, -30, and -43 and a significant increase in gap-junction-mediated dye transfer. Transient expression of mutant Cx26(D50N) in PCECs resulted in cytoplasmic accumulation and lack of gap junction plaque formation and was not altered by coexpression of wild-type (wt)Cx26 or -30. CONCLUSIONS: Gap junction communication in the human corneal epithelium is mediated by Cx26, -30, -31.1, and -43. Poorly differentiated PCECs are uncoupled, and Ca(2+) induced differentiation is associated with an upregulation of connexin expression and intercellular communication. The transfection experiments suggest that KIDS Cx26(D50N) impairs intracellular formation and transport of connexons formed by Cx26 and -30, consistent with a dominant negative effect.

Adult↗

Human bradykinin B2 receptor: nucleotide sequence analysis and assignment to chromosome 14.

Functional cDNA clones for human bradykinin B2 receptor were isolated from uterus RNA by a polymerase chain reaction (PCR)-based method and by screening a human cosmid library with rat bradykinin B2 receptor probe. We isolated several overlapping clones from the cosmid library, each of which encodes the entire protein coding sequence. The human bradykinin B2 receptor gene codes for a 364-amino-acid protein with a molecular mass of 41,442 Da that is highly homologous to rat bradykinin B2 receptor cDNA (81%). The entire human cDNA sequence was cloned into an expression vector and mRNA was synthesised by in vitro transcription. Applications of bradykinin caused membrane current responses in Xenopus oocytes injected with the in vitro-synthesized mRNA. Preincubation with the potent B2 antagonist, HOE140, prevented this response. The genomic clone is intronless, and we have identified an upstream promoter region and a downstream polyadenylation signal. The human bradykinin B2 receptor gene has been mapped to chromosome 14 using PCR to specifically amplify DNA from somatic cell hybrids.

Amino Acid Sequence↗

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

Artifacts↗

Serological response to HPV16 in CIN-III and cervical cancer patients. Case-control studies in Spain and Colombia.

This study evaluates the association of antibodies against HPV-16-derived peptides with cervical cancer and estimates the sensitivity and specificity of the serological assays in relation to HPV DNA detection in cervical cells by PCR. Study subjects were derived from 4 case-control studies carried out in Spain and Colombia. Sera from 544 cases of CIN III and invasive cancer and of 543 age-matched controls were tested for antibodies to 5 peptides derived from E2, E7 (3 partially overlapping frames of HPV 16 denoted E7/ 1, E7/2, E7/3) and L2 open reading frames of HPV 16. HPV DNA was detected using a L1-PCR based method. Among cancer controls, antibody response to E2 and E7/1, E7/2, E7/3 was higher in Colombia (22.5%,7.2%,11.7%,12.6% respectively) than in Spain (17.1 %, 4.7%, 5.9%, 5.9%). E7 antibodies were related to stage, particularly in CIN III vs. invasive stages and less markedly within invasive stages. Detection of antibodies to the E7/1 was associated to CIN III (OR = 1.8). The risk of invasive cervical cancer was increased among those with antibodies to E2 (OR = 2.2), to E7/1 (OR = 4.2), to E7/2 (OR = 4.3), and to E7/3 (OR = 2.5). Presence of antibodies to all the 3 E7 peptides increased the risk of CIN III (OR = 5.6) and that of invasive cancer (OR = 17.5). High levels of antibodies to E7/1 or E7/2 or E7/3 increased the risk of invasive cervical cancer (OR for high levels of antibodies vs. negatives to E7/1 OR = 22.6; E7/2 OR = 7.5, E7/3 OR = 3.4). In the present analysis, antibodies to L2 were not associated with either CIN III or cervical cancer. Serological markers of HPV 16 detected less than half of the HPV-16-DNA-positive cases. It is concluded that antibodies to E2 and particularly E7 antigens are strongly associated with cervical cancer. Antibodies to E7 seem to be a moderate marker of tumor burden.

Adult↗

Recombining overlapping BACs into a single larger BAC.

BACKGROUND: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. RESULTS: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. CONCLUSION: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones.

Chromosomes, Artificial, Bacterial↗

Homogeneous polymerase chain reaction nucleobase quenching assay to detect the 1-kbp deletion in CLN3 that causes Batten disease.

Batten disease is an autosomal recessive disorder also known as juvenile neuronal ceroid lipofuscinosis. The most common mutation for this disease is an approximately 1-kbp deletion in the CLN3 gene, which accounts for about 80 to 85% of the mutation load. We developed a rapid assay for this mutation using the PCR to produce amplicons that are detected by nucleobase quenching of the fluorescent signal from a probe labeled with a fluorescent dye. The probe overlaps the deletion breakpoint and is completely base paired to the mutant amplicon. However, three bases at the 5' end of the probe do not base pair with the wild-type amplicon. The alleles are distinguished by the different melting temperatures of the probe amplicon hybrids. Comparison of this new method with an allele-specific PCR and gel electrophoresis-based method showed 100% concordance in determination of the genotype for 30 specimens (11 homozygous mutant, 8 heterozygotes, and 11 homozygous normal). PCR followed by allele-specific melting curve analysis using nucleobase quenching has utility as a rapid method for detection of the most common mutation that causes Batten disease.

Female↗

Chromosomal localization of the human gene for annexin V (placental anticoagulant protein I) to 4q26----q28.

Annexin V is a member of a new family of calcium-dependent phospholipid-binding proteins. It has been previously isolated as placental anticoagulant protein I, inhibitor of blood coagulation, vascular anticoagulant-alpha, endonexin II, lipocortin V, placental protein 4, and anchorin CII. The human gene encoding annexin V (ANX5) was localized to 4q26----q28 by in situ hybridization with a cDNA probe and polymerase chain-reaction (PCR) analysis of a human x hamster hybrid cell panel. The regional localization to 4q26----q28 was supported by Southern-blot analysis of a human cell line with a deletion in 4q23----q27. This localization overlaps but differs slightly from the previous assignment of ANX5 to 4q28----q32. Digestion with PvuII and TaqI identified polymorphisms at the ANX5 locus; the PvuII polymorphism could also be detected by PCR analysis.

Animals↗

Glycogen storage disease type Ib: structural and mutational analysis of the microsomal glucose-6-phosphate transporter gene.

Glycogen storage disease type Ib is caused by a mutation in the gene encoding microsomal glucose-6-phosphate (G6P) transporter. We determined the exon/intron organization of the G6P transporter gene. Four overlapping genomic fragments containing the entire coding region of the gene were amplified by polymerase chain reaction (PCR) using exonic primers, and their nucleotide sequences were determined. The gene spans 4.5 kb and has eight exons. All exon/intron boundaries adhered to the canonical AG/GT rule. We then designed eight pairs of PCR primers to amplify all coding exons for a mutational analysis and studied five Japanese patients with the disease. Two novel homozygous mutations were identified in two families: a three-base deletion (delV235) in exon 2 in a consanguineous family and a splicing mutation (IVS7+1G-->T) in intron 7 in a nonconsanguineous family. Patient 3 was a compound heterozygote of W118R and IVS1+1G-->A, both of which we previously identified [Kure et al., 1998: Biochem Biophys Res Commun 248:426-431]. Patients 4 and 5 were homozygotes of W118R. Including our previous study, we found a total of ten W118R alleles in nine Japanese patients. The results support our previous suggestion that W118R is prevalent among Japanese patients. The genomic sequence data and mutation spectrum obtained from the Japanese patients will facilitate genetic diagnosis of glycogen storage disease type Ib.

Antiporters↗

Neuroblastoma tumors with favorable and unfavorable outcomes: Significant differences in mRNA expression of genes mapped at 1p36.2.

The distal part of 1p is frequently deleted in aggressive neuroblastoma, and the region is believed to harbor one or more tumor suppressor genes relevant to tumor development. To analyze differences among neuroblastoma tumors, an expression profile was established for the genes mapped within a previously described shortest region of overlap of deletions at 1p36.2. The gene expression levels were quantified by TaqMan real-time (RT)-PCR for 30 transcripts using 55 primary neuroblastoma tumors. Here we report on a significant decrease in gene expression of the genes RERE, PIK3CD, LZIC, PGD, and PEX14 and an increase of SLC2A5 when comparing tumors of favorable biology to Stage 4 neuroblastomas. When comparing 1p-deleted tumors of all stages to tumors with an intact 1p, a significant difference at gene-by-gene level in TNFRSF9, RERE, PIK3CD, CLSTN1, CTNNBIP1, and CASZ1 was detected. A complete loss of expression could not be seen for any single gene analyzed. Several of the genes with diminished expression in unfavorable or 1p-deleted tumors have functions that could contribute to tumor development. It is also possible that a combination of lowly expressed genes at 1p, rather than one single classical tumor suppressor gene, causes the unfavorable outcome associated with 1p-deletion in neuroblastoma.

Chromosome Deletion↗

Structure and organization of the human glucose phosphate isomerase gene (GPI).

Two overlapping yeast artificial chromosome clones containing the human glucose 6-phosphate isomerase gene (GPI) have been isolated. PCR and direct sequencing were used to determine the exon/intron structure of the gene. The gene spans in excess of 40 kb and consists of 18 exons ranging in size from 44 to 153 bp. All splice sites conform to the GT/AG rule.

Animals↗

Reconstitution of an episomal mouse aprt gene as a consequence of recombination.

When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt- L cells, the cells are rendered Aprt+ and the aprt gene persists as an episome. Cotransfection with two BPV vectors, one containing the 5' half of the aprt gene and the other the 3' half of the gene, that share about 300 bp of common sequence in intron 2, produces Aprt+ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic SmaI fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5' half or 3' half of aprt were transfected into Aprt- L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt+ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.

Adenine Phosphoribosyltransferase↗

Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides.

This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally. The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker. An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns. The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively. The atypical Kozak sequence is identical to the one used in R. oryzae in positions -1 to -4. Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose. In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence.

Amino Acid Sequence↗

Molecular cloning of the branched-chain alpha-keto acid dehydrogenase kinase and the CoA-dependent methylmalonate semialdehyde dehydrogenase.

The complete amino acid sequence of rat liver CoA-dependent methylmalonate semialdehyde dehydrogenase, the enzyme responsible for the oxidative decarboxylation of malonate- and methylmalonate semialdehydes to acetyl- and propionyl-CoA in the distal portions of the valine and pyrimidine catabolic pathways, has been deduced from overlapping cDNAs obtained by screening a lambda gt11 library with nondegenerate oligonucleotide probes synthesized according to PCR-amplified portions coding for the N-terminal amino acid sequence of the enzyme. Although unique because of its requirement for coenzyme A, the methylmalonate semialdehyde dehydrogenase clearly belongs to the aldehyde dehydrogenase superfamily of enzymes. Quantitation of mRNA and protein levels indicates tissue-specific expression of methylmalonate semialdehyde dehydrogenase. A large increase in expression of methylmalonate semialdehyde dehydrogenase occurs during 3T3-L1 preadipocyte differentiation into adipocytes. The complete amino acid sequence of rat liver branched-chain alpha-ketoacid dehydrogenase kinase, the enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, was deduced from a cDNA cloned by a procedure similar to that described above for the methylmalonate semialdehyde dehydrogenase. Expression of the cDNA in E. coli yielded a protein that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex. Very little sequence similarity between branched-chain alpha-ketoacid dehydrogenase kinase and other eukaryotic protein kinases could be identified. However, a high degree of similarity within subdomains characteristic of prokaryotic histidine protein kinases was apparent. Thus, this first mitochondrial protein kinase to be cloned appears closer, evolutionarily, to the prokaryotic histidine protein kinases than eukaryotic ser/thr protein kinases.

3T3 Cells↗

A YAC-, P1-, and cosmid-based physical map of the BRCA1 region on chromosome 17q21.

A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated. The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap. The majority of the interval has also been converted to a cosmid contig. Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones. This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al. (1995), Osborne-Lawrence et al. (1995), and Friedman et al. (1995).

BRCA1 Protein↗

Global gene repression in hepatocellular carcinoma and fetal liver, and suppression of dudulin-2 mRNA as a possible marker for the cirrhosis-to-tumor transition.

BACKGROUND/AIMS: Whether the transcriptional reprogramming process induced by hepatocellular carcinoma recapitulates that of the developing liver is at present unclear. METHODS: With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22-24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR. RESULTS: Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis. CONCLUSIONS: Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.

Aged↗

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum (SR) Ca(2+)-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with approximately 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five 'stalk' domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca(2+)-ATPases is discussed.

Amino Acid Sequence↗

Spectrophotometric quantitative determination of cilazapril and hydrochlorothiazide in tablets by chemometric methods.

Four chemometric methods were applied to simultaneous determination of cilazapril and hydrochlorothiazide in tablets. Classical least-square (CLS), inverse least-square (ILS), principal component regression (PCR) and partial least-squares (PLS) methods do not need any priori graphical treatment of the overlapping spectra of two drugs in a mixture. For all chemometric calibrations a concentration set of the random mixture consisting of the two drugs in 0.1 M HCI and methanol (1:1) was prepared. The absorbance data in the UV-Vis spectra were measured for the 15 wavelength points (from 222 to 276 nm) in the spectral region 210-290 nm considering the intervals of deltalambda = 4 nm. The calibration of the investigated methods involves only absorbance and concentration data matrices. The developed calibrations were tested for the synthetic mixtures consisting of two drugs and using the Maple V software the chemometric calculations were performed. The results of the methods were compared each other as well as with HPLC method and a good agreement was found.

Algorithms↗