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Genomic organization of the gene and a related pseudogene for a human folate binding protein.

An unprocessed pseudogene which is 90% homologous with the cDNA encoding a folate binding protein in KB cells has been cloned from a human genomic library. This pseudogene contains TGA stop codons, base deletions and substitutions and lacks a 5' region. The size of the exons and the intron-exon sites are almost identical to the organization of the gene encoding this protein which has now been characterized from genomic DNA using the polymerase chain reaction with selected primers to the cDNA.

Amino Acid Sequence↗

[Construction of DNA-fragments' libraries with complete genomes of different variola strains].

Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.

DNA, Viral↗

Structure and regulation of a polymorphic gene encoding folate receptor type gamma/gamma'.

The human folate receptor (hFR) type gamma and gamma' are constitutively secreted proteins that are expressed primarily in hematopoietic tissues and are potential serum markers for certain hematopoietic malignancies. hFR-gamma' is a variant of hFR-gamma with a two base deletion in its cDNA resulting in a truncated polypeptide. The gene encoding hFR-gamma' was isolated from a placental genomic library. The gene has five exons, four introns and a 5' flanking sequence which contains multiple putative regulatory elements. From RNase protection assay and RACE analysis, the major site of transcriptional initiation was identified at -56 nt. Systematically deleted fragments in the 5' region of the genomic DNA of FR-gamma' were ligated into the PGL3Basic plasmid and the reporter luciferase activity was assayed in cell lysates from transiently transfected NIH3T3 cells. From those results, putative positive and negative regulatory regions in the 5' flanking sequences were noted, and a TATA-less proximal promoter was located between -206 and -22 nt. Gel mobility shift and supershift analyses as well as mutagenesis experiments revealed that Sp1 and ets binding elements in the proximal promoter region confer transcriptional activity. From partial sequencing of genomic DNA, genomic Southern blots, RACE analysis and RNase protection assays, it appears that hFR-gamma shares the gene organization of hFR-gamma'. The results of the analysis of genomic DNA in spleen tissues from several individuals, were consistent with the interpretation that hFR-gamma and hFR-gamma' are encoded by a polymorphic gene.

Amino Acid Sequence↗

Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.).

Repetitive DNA elements representing 60-70% of the total repetitive DNA in tetraploid cotton (Gossypium barbadense L.) and comprising 30-36% of the tetraploid cotton genome were isolated from a genomic library of DNA digested with a mixture of four blunt-end cutting restriction enzymes. A total of 313 clones putatively containing nuclear repetitive sequences were classified into 1103 families, based on cross hybridization and Southern blot analysis. The 103 families were characterized in terms of genome organization, methylation pattern, abundance, and DNA variation. As in many other eukaryotic genomes, interspersed repetitive elements are the most abundant class of repetitive DNA in the cotton genome. Paucity of tandem repeat families with high copy numbers (>10(4)) may be a unique feature of the cotton genome as compared with other higher plant genomes. Interspersed repeats tend to be methylated, while tandem repeats seem to be largely unmethylated in the cotton genome. Minimal variation in repertoire and overall copy number of repetitive DNA elements among different tetraploid cotton species is consistent with the hypothesis of a relatively recent origin of tetraploid cottons.

Base Sequence↗

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

Aedes↗

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.).

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.

Amino Acid Sequence↗

The leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like Loci.

The innate and adaptive immune systems of vertebrates possess complementary, but intertwined functions within immune responses. Receptors of the mammalian innate immune system play an essential role in the detection of infected or transformed cells and are vital for the initiation and regulation of a full adaptive immune response. The genes for several of these receptors are clustered within the leukocyte receptor complex (LRC). The purpose of this study was to carry out a detailed analysis of the chicken (Gallus gallus domesticus) LRC. Bacterial artificial chromosomes containing genes related to mammalian leukocyte immunoglobulin-like receptors were identified in a chicken genomic library and shown to map to a single microchromosome. Sequencing revealed 103 chicken immunoglobulin-like receptor (CHIR) loci (22 inhibitory, 25 activating, 15 bifunctional, and 41 pseudogenes). A very complex splicing pattern was found using transcript analyses and seven hypervariable regions were detected in the external CHIR domains. Phylogenetic and genomic analysis showed that CHIR genes evolved mainly by block duplications from an ancestral inhibitory receptor locus, with transformation into activating receptors occurring more than once. Evolutionary selection pressure has led not only to an exceptional expansion of the CHIR cluster but also to a dramatic diversification of CHIR loci and haplotypes. This indicates that CHIRs have the potential to complement the adaptive immune system in fighting pathogens.

Alternative Splicing↗

New members of the 3 beta-hydroxysteroid dehydrogenase gene family.

Several bands of hydridization are detected when southern blots of human genomic DNA are proved with cDNA of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly, primer designed to amplify 3 beta-HSD type I and II genes were found on occasion to amplify DNA products of appropriate length but which were resolved as distinct sequences by denaturing gradient gel electrophoresis (DGGE). Five of these novel bands were cloned and their sequences were found to be closely related to 3 beta-HSD types I and II. Secondly, 57 genomic clones were selected from two lambda genomic libraries by hybridization with exonic probes of 3 beta -HSD type I. These were screened for novel members of the gene family by pcr amplification using various combinations of PCR primers to the type I and II genes, particularly those primers that previously amplified novel PCR products from genomic DNA. Amplification products from (lambda) clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3 beta-HSD gene family were found, one of which locates to the 3 beta -HSD type I and II gene cluster on 1p13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from (lambda) clones to search for novel members of the gene family.

3-Hydroxysteroid Dehydrogenases↗

Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene.

Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors.

Amino Acid Sequence↗

Transient expression assay for qualitative assessment of gene expression by fowlpox virus.

A transient expression assay for fowlpox virus (FPV) was developed to assess the feasibility of using heterologous promoters in FPV and to qualitatively determine relative promoter strength. A transient expression system for FPV has not been reported, and various methods used for transient expression in vaccinia-virus-infected cells produced negative results when used with FPV. Here a successful method for transient expression of E. coli beta-galactosidase in FPV-infected chick embryo fibroblasts is reported. This transient expression assay has been developed to qualitatively assess promoter recognition and gene expression by FPV. It should also prove useful in the identification of promoters from the FPV genomic library and in testing the accuracy of chimeric promoter-gene constructs.

Animals↗

A human DNA segment encompassing leucine and methionine tRNA pseudogenes localized on chromosome 6.

A human genomic clone, designated LHtlm8, that strongly hybridized to a mammalian leucine tRNA(IAG) probe, was found to encompass a pair of tRNA pseudogenes that are transcribed in a homologous cell extract. A leucine tRNA(AAG) pseudogene (TRLP1) is 2.1-kb upstream and of opposite polarity to a methionine elongator tRNA(CAU) pseudogene (TRMEP1). TRLP1 has three nucleotide variations (97% identity) from its cognate leucine tRNA(IAG), while TRMEP1 has a 78% identity with its cognate tRNA. Similar to a number of other eukaryotic tRNA pseudogenes, presumptive precursor tRNA transcripts are generated from the two pseudogenes in vitro, but possibly due to their aberrant and unstable secondary and tertiary structures, no detectable mature tRNA products are observed. The two tRNA pseudogenes are encompassed within a 9.6-kb EcoRI fragment that has been assigned to the chromosomal locus, 6pter-q13, by Southern blot hybridization of human-rodent somatic cell hybrid DNAs with probes derived from the cloned tRNA pseudogenes and flanking sequences. A 4.4-kb EcoRI fragment also harbored in clone LHtlm8 was mapped to human chromosome 11, suggesting that the two EcoRI fragments were inadvertantly ligated together during construction of the genomic library.

Animals↗

A rat histone H2B pseudogene is closely associated with the histone H1d gene.

A 9 kb EcoRI restriction fragment was isolated from a recombinant phage out of a rat genomic library. This DNA fragment contains a rat H1d histone gene, its flanking sequences and a H2B histone pseudogene closely associated with the H1d gene. A comparison of the H2B pseudogene with human H2B genes flanking regions reveals sequence homologies to a human H2B histone gene (Albig, W. et al. (1991) Genomics 10, 940-948).

Amino Acid Sequence↗

Molecular analysis of the genes mediating Salmonella invasion.

To identify invasion determinants, a genomic library of Salmonella typhimurium was cloned into a cosmid vector, pLA2917. A clone, pSI623 which was invasive for HEp-2 and Henle-407 epithelial cells, was subcloned into a plasmid vector, pGEM-7Z to define the invasion genes. The subclone, pSV6235 containing a 4.5 kbp fragment of the Salmonella genomic region, was highly invasive for HEp-2 and Henle-407 cells, compared with other subclones whose Salmonella genomic regions are 7.4, 6.3, 5.5 and 1.4 kbp, respectively. This study reflects that the invasion efficiency of the host strain, Escherichia coli to HEp-2 and Henle-407 cell lines was significantly increased by the introduction of the genomic region of the virulent S. typhimurium. Restriction enzyme analysis showed that there was a single PstI site on the 27 kbp of Salmonella genomic region of pSI623, and no PstI site was found on the 4.5 kbp of genomic region of pSV6235. This finding suggests that the invasion related genes different from the inv and spa gene clusters which were identified in S. typhimurium, may exist in genomic DNA of S. typhimurium.

Cell Line↗

The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift.

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.

Adhesins, Bacterial↗

Differential diagnosis of Taenia saginata and Taenia solium infection by PCR.

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1, 272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.

Animals↗

Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.

A trans-acting gene required for late viral gene expression in transient expression assays was identified in the genome of Autographa californica nuclear polyhedrosis virus. A genomic library of A. californica nuclear polyhedrosis virus DNA lacking a clone spanning the region from 43 to 48 map units (mu) was unable to activate gene expression from a reporter plasmid when the reporter gene was driven by a baculovirus late or very late promoter in transient expression assays. The genomic region responsible for activating reporter gene expression was further mapped to 43.4 to 45.2 mu of the viral genome. The nucleotide sequence of this region was determined and shown to contain several small open reading frames (ORFs) and one major ORF, named lef-3, for late expression factor 3. The lef-3 ORF was predicted to encode a polypeptide of 385 amino acids and with a molecular mass of 44,529 daltons. No homolog to the lef-3 ORF was found in existing data bases. Further analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in the assays used. The temporal regulation of lef-3 transcription was studied by Northern (RNA) blot hybridization; lef-3 was found to be an early gene that was transcribed primarily as a 2.0-kb mRNA. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG codon and terminated near a polyadenylation signal, 130 bp downstream of the last codon of the lef-3 ORF.

Amino Acid Sequence↗

Genome organization of repetitive elements in the rodent, Peromyscus leucopus.

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)n was present in 87% of the clones, (CT)n was present in 59% of the clones, and (AT)n and (GC)n each was represented in our sample by a single clone (0.05%). (TCC)n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.

Animals↗

Molecular cloning and sequencing of the murine gastrin gene.

Gastrin is a peptide hormone important in acid regulation, growth of enterochromaffin cells of the oxyntic mucosa, and smooth muscle contractility. We isolated a genomic clone of gastrin from a SV-129 murine genomic library. The murine gastrin gene contains an open reading frame of 101 amino acids. The deduced amino acid sequence has 91.1% homology with the rat and 67.3% homology with the human analogue. Southern blot analysis of the murine gastrin clone gave the same restriction pattern as that seen from mouse kidney genomic DNA, consistent with this being a single copy of the gastrin gene in the mouse genome. Transfection studies demonstrated that the murine gastrin gene expression is stimulated by epidermal growth factor to the same extent as the human gastrin gene.

Amino Acid Sequence↗