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PubMed1997

Construction of a 5-Mb YAC contig from the putative 10q25 tumor-suppressor region for glioblastomas.

During the final step of the malignant progression to glioblastoma multiforme (GBM), the most frequent and malignant of primary brain tumors, more than 90% of the cases exhibit loss of genetic material on chromosome 10. We previously identified a 4-cM deletion interval in the 10q24-qter region that is common to all the GBM we have examined. A contig of 20 YACs spanning the 5 Mb of chromosomal DNA in the region has been assembled. Overlaps between YACs have been verified by STS content, fingerprinting analysis, and/or Alu-Alu PCR. The contig contains 17 known microsatellite markers, 15 new STSs derived from the insert ends of YACs, 9 ESTs, and 11 others STSs, for a total of 52 STSs (average marker density 1/100 kb). The physical map of this region will facilitate the search for a candidate tumor-suppressor gene(s) that is inactivated during the information of GBM.

Base Sequence↗
PubMed1999

Identification and characterization of the molecular lesion causing mucopolysaccharidosis type I in cats.

Mucopolysaccharidosis Type I (MPS I) is the lysosomal storage disease caused by the deficient activity of alpha-L-iduronidase (IDUA). In man, MPS I can occur in severe, mild, or intermediate forms known as the Hurler, Scheie, or Hurler/Scheie syndromes, respectively. MPS I also has been described in cats, dogs, and mice. This manuscript reports the identification and characterization of the mutation causing MPS I in cats. To obtain wild-type feline IDUA cDNAs, two PCR-based strategies were used. PCR primers were constructed from a conserved region of the published human and dog sequences and used to amplify a 224-bp IDUA fragment from normal cat genomic DNA. This fragment was then used to screen a feline uterus cDNA library. PCR also was used to directly amplify IDUA fragments from the same cDNA library. Two overlapping feline IDUA cDNAs encoding 466 amino acid residues of the feline IDUA polypeptide ( approximately 85% of the mature protein based on comparison to the human, dog, and mouse sequences) were obtained by these strategies. To identify the mutation causing MPS I in cats, DNA sequencing was carried out on the corresponding IDUA region from several affected animals. A 3-bp deletion was found on both IDUA alleles in each of the MPS I animals, predicting the deletion of a single aspartate residue from the feline IDUA polypeptide. To confirm the authenticity of this mutation, heteroduplex, SSCP, and transient expression studies were carried out. Over 100 animals from the MPS I colony were screened for the presence of the mutation by heteroduplex and SSCP analyses-in all cases the presence of the 3-bp deletion was 100% concordant with the disease phenotype. For transient expression studies, the two partial, overlapping feline cDNAs were combined and joined in-frame to the 5' end of the canine IDUA cDNA. This wild-type, hybrid cDNA expressed IDUA activity up to sixfold over endogenous levels after transfection into COS-1 cells. A modified full-length IDUA cDNA containing the 3-bp deletion did not express IDUA activity in a transient expression system, providing proof that this lesion was the cause of feline MPS I.

Animals↗
PubMed1994

Cloning and sequencing of the Lactococcus lactis subsp. lactis dnaK gene using a PCR-based approach.

The coding region for the dnaK gene from Lactococcus lactis subsp. lactis LM0230 was isolated and sequenced. An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK. This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene. The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions. The inverse PCR products were then cloned and partially sequenced. The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein. Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.

Amino Acid Sequence↗
PubMed1997

Delimitation of a critical tumour suppressor region at distal 1p in neuroblastoma tumours.

We analysed DNA from 68 neuroblastoma tumours for loss of heterozygosity (LOH) on the distal chromosome 1p (1p-LOH) using PCR-based DNA polymorphisms. Fifteen tumours (22%) displayed 1p-LOH. The shortest region of overlap (SRO) for the deletions was defined proximally by marker D1S244 and distally by marker D1S80. The CDC2L1 locus, located on chromosome 1p36, has been put forward as a neuroblastoma tumour suppressor. We analysed coding regions of the CDC2L1 gene in a subset of aggressive neuroblastoma tumours with known allelic loss for different 1p-markers. Single-stranded conformation polymorphism, heteroduplex and sequencing analysis of tumour DNA did not reveal any significant changes in the coding region. Using a DNA sequence polymorphism, we showed, in a primary tumour with an interstitial allelic deletion, that this tumour had both alleles of the CDC2L1 locus retained in the tumour. Thus, we showed that the neuroblastoma tumour suppressor critical region on 1p in our material is defined by loci D1S244 and D1S80 and that the CDC2L1 locus is distal to the critical region.

Child↗
PubMed2003

Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major.

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.

Amino Acid Sequence↗
PubMed2006

New insights into the biology of cytokinin degradation.

A survey of recent results is presented concerning the role of cytokinin degradation in plants, which is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. An overview of Arabidopsis CKX gene expression suggests that their differential regulation by biotic and abiotic factors contributes significantly to functional specification. Here, we show using reporter gene and semiquantitative RT-PCR analyses regulation of individual CKX genes by cytokinin, auxin, ABA, and phosphate starvation. Partially overlapping expression domains of CKX genes and cytokinin-synthesizing IPT genes in meristematic tissues and endo-reduplicating cells lend support for a locally restricted function of cytokinin. On the other hand, their expression in vascular tissue suggests a function in controlling transported cytokinin. Recent studies led to a model for the biochemical reaction mechanism of CKX-mediated catalysis, which was refined on the basis of the three-dimensional enzyme structure. Last but not least, the developmental functions of CKX enzymes are addressed. The recent identification of the rice OSCKX2 gene as an important novel breeding tool is highlighted. Together the results corroborate the relevance of metabolic control in determining cytokinin activity.

Arabidopsis↗
PubMed1994

Structure and expression of the hairless gene of mice.

The hairless mutation of mice was caused by insertion of a murine leukemia virus. Starting with sequences flanking the provirus, a series of overlapping clones surrounding the viral integration site were obtained. By using a combination of sequencing, PCR, and exon-trapping techniques, the hairless gene was identified. It encodes a predicted protein of 1182 amino acids, including a potential zinc-finger domain. The expression patterns of the gene closely reflect the phenotype of animals carrying the hairless mutation.

Amino Acid Sequence↗
PubMed1996

Sequence of the 11 beta-hydroxylase gene from the Cape baboon (Papio ursinus).

We investigated the nucleotide sequence of the steroid 11 beta-hydroxylase gene (CYP11B1) from the Cape baboon (Papio ursinus). Six primers, previously used in studies on human CYP11B1, were utilised to amplify three overlapping fragments (A, B and C) of the baboon CYP11B1 by the polymerase chain reaction (PCR). Sequence analysis of the three fragments yielded the sequence of all the exons of baboon CYP11B1. The open reading frame of 1509 bases shows 57 nucleotide exchanges when compared to the human resulting in 18 amino acid substitutions. For eight of these exchanges we found the amino acid which is common for human aldosterone synthase at the corresponding position. Most of the remaining 10 amino acid substitutions were conservative. Eight of the substitutions were located in the first four exons with a cluster in the second half of exon 3. One substitution was in exon 5 (F280L) and the 10th was C494F at the end of the protein.

Animals↗
PubMed1996

Genomic structure and nucleotide sequence analysis of a new HIV type 1 subtype A strain from Nigeria.

We have isolated and characterized a new HIV-1 variant (HIV-1IbNg) from the peripheral blood mononuclear cells (PBMCs) of an inhabitant of Nigeria. This virus is highly cytopathic to PBMCs in culture, replicates in primary human T cells and macrophages/monocytes as well as in established human T cell and monocytic cell lines (i.e., it has a wide host range), and it does not induce syncytia in MT-2 cells.1 Because of these unusual infectivity parameters in vitro, we analyzed the genetic structure of the entire genome. Using cytoplasmic RNA from HIV-1IbNg-infected PBMCs, five overlapping DNA fragments were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and cloned into pBluescript II SK(+). DNA sequencing of these fragments indicated that the entire HIV-1IbNg genome consisted of 9201 nucleotides and phylogenetic analysis of its env gene sequence revealed that this virus clustered with HIV-1 strains belonging to clade "A". In this article we present several genetic features unique to this virus, including (1) the presence of a 16-bp insert within the primer-binding site, (2) a large Rev open reading frame, (3) a Rev-responsive element that is predicted to form a different secondary structure than described for clade "B" viruses, (4) the potential to encode a heavily glycosylated Env protein, and (5) a frameshift, resulting in a stop codon, in the tat gene. This represents the first detailed analysis of the genetic structure of an HIV-1 strain from Nigeria.

Adult↗
PubMed2002

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

Alleles↗
PubMed2004

An efficient one-step site-directed and site-saturation mutagenesis protocol.

We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.

DNA Primers↗
PubMed2006

Report of two fatal cases of Mycobacterium mucogenicum central nervous system infection in immunocompetent patients.

Neurological infections due to rapidly growing mycobacteria (RGM) have rarely been reported. We recently investigated two unrelated immunocompetent patients, one with community-acquired lymphocytic meningitis and the other with cerebral thrombophlebitis. Mycobacterium mucogenicum was isolated in pure culture and detected by PCR sequencing of cerebrospinal fluid samples. Both patients eventually died. The two isolates exhibited an overlapping antimicrobial susceptibility pattern. They were susceptible in vitro to tetracyclines, macrolides, quinolones, amikacin, imipenem, cefoxitin, and trimethoprim-sulfamethoxazole and resistant to ceftriaxone. They shared 100% 16S rRNA gene sequence similarity with M. mucogenicum ATCC 49650T over 1,482 bp. Their partial rpoB sequences shared 97.8% and 98.1% similarity with M. mucogenicum ATCC 49650T, suggesting that the two isolates were representative of two sequevars of M. mucogenicum species. This case report should make clinicians aware that M. mucogenicum, an RGM frequently isolated from tap water or from respiratory specimens and mostly without clinical significance, can even be encountered in the central nervous system of immunocompetent patients.

Adult↗
PubMed2001

Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter.

Transcription of the human inducible nitric oxide synthase (iNOS) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5'-flanking region. Data showed both constitutive and inducible HS sites in an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near -5 kb containing both constitutive and cytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells.

Base Sequence↗
PubMed2025

Transcriptomics-based exploration of ubiquitination-related biomarkers and potential molecular mechanisms in laryngeal squamous cell carcinoma.

BACKGROUND: One of the most common and prevalent cancers is laryngeal squamous cell carcinoma (LSCC), which poses a great threat to the life and health of the patient. Nonetheless, it has been demonstrated that ubiquitination is crucial for the development and course of LSCC. Therefore, it is particularly important to identify biomarkers for ubiquitination-related genes (UbRGs) in LSCC. METHODS: Differentially expressed genes (DEGs) in the LSCC versus controls were obtained by differential expression analysis. Also, key modular genes associated with LSCC were obtained using weighted gene co-expression network analysis (WGCNA). Next, DEGs, key module genes, and UbRGs were taken to intersect to obtain candidate genes. And then machine algorithms were to screen potential biomarkers, further their diagnostic value were analyzed and validated. Then, therapeutic agents for biomarkers were predict. In addition, the regulatory networks of the biomarkers were mapped. The expression levels of biomarkers were detected in clinical samples using reverse transcription-quantitative PCR (RT-qPCR). RESULTS: A total of eight candidate genes were acquired by the overlap 1,911 DEGs, the key modular genes of WGCNA, and 1,393 UbRGs. A sum of four biomarkers (WDR54, KAT2B, NBEAL2 and LNX1) were identified by two machine learning, then these four biomarkers were validated in GSE127165 and the expression trend was consistent with TCGA-LSCC, they were recorded as biomarkers. Moreover, the accuracy of the biomarkers in predicting clinical aspects of LSCC was confirmed by the receiver operating characteristic (ROC) curves. Subsequently, cancers such as malignant neoplasms, colorectal cancers, tumors, and primary malignant neoplasms were significantly associated with the biomarkers, which further suggests that these four biomarkers were strongly associated with cancer. Meanwhile, the drugs garcinol, cocaine, and triazolam, among others, used for LSCC treatment were predicted. Finally, transcription factors (TFs) (BRD4, MYC, AR, and CTCF) were predicted to regulate the biomarkers. RT-qPCR assays illustrated that the expression trends of KAT2B, LNX1 and NBEAL2 remained consistent with the dataset. CONCLUSION: The identification of four biomarkers (WDR54, KAT2B, NBEAL2 and LNX1) associated with UbRGs could ultimately serve as a predictive clinical diagnosis of LSCC and provide insight into the molecular mechanisms of LSCC.

Humans↗
PubMed2005

Detection of common and private mutations in the COL6A1 gene of patients with Bethlem myopathy.

BACKGROUND: Dominant mutations in COL6A1, COL6A2, and COL6A3, the three genes encoding collagen type VI, a ubiquitous extracellular matrix protein, are associated with Bethlem myopathy (BM) and Ullrich scleroatonic muscular dystrophy. METHODS: The authors devised a method to screen the entire coding sequence of the three genes by reverse transcriptase-PCR amplification of total RNA from skin fibroblasts and direct sequencing of the resulting 25 overlapping cDNA fragments covering 107 exons. RESULTS: Four splicing and four missense mutations were identified in 16 patients with BM, six of which are novel mutations in COL6A1. Both common and private mutations are localized in the alpha1 (VI) chain between the regions corresponding to the 3' end of the NH2-globular domain and the 5' end of the triple helix, encoded by exons 3 through 14. CONCLUSIONS: The clustering of the mutations in a relatively narrow area of the three collagen type VI chains in patients with Bethlem myopathy (BM) suggests that mutations in different regions could result in different phenotypes or in no phenotype at all. Moreover, the detection of mutations in only 60% of the patients suggests the existence of at least another gene associated with BM. The authors propose the direct sequencing of COL6 cDNAs as the first mutation screening analysis in BM, given the high number of exon-skipping events.

Adolescent↗
PubMed2001

[Nucleotide sequence of the noncoding regions of measles virus stain CC-47 and comparison with other measles viruses].

BACKGROUND: To determine the nucleotide sequence of noncoding regions of Measles virus strain Changchun-47 (CC-47) and to compare them with other measles virus for revealing some vaccine-related information. METHODS: Six overlapped fragments, that covers complete genome of CC-47 were amplified by using RT-PCR, all of the amplified fragments have been cloned and sequenced. RESULTS: Seven noncoding regions lie in the genome sequence of CC-47 separated by six structure genes. The noncoding regions of CC-47 contain 1791 nucleotides totally. Comparing of the noncoding regions of CC-47 with other wild type strains and five Edmonston-derived vaccine strains, four nucleotide substitutions were shared by nearly all vaccine strains. Two of these were in the genomic 3 terminal transcriptional control region: position 26 (U->G), 42 (U->G); the other were in the F mRNA 5 -untranslated region of M/F intergenic region. These site substitutions may influence the efficiency of mRNA synthesis, processing, and translation, as wel l as genome replication and encapsidation. CONCLUSIONS: The nucleotide substitutions shared by different genotype measles virus vaccine strains may be related to virus growth in semipermissive cell or process of attenuation, which provide useful data for molecular epidemiology of measles virus. The nucleotide substitutions for attenuation were involved in several regions other than one definite region.

3' Untranslated Regions↗
PubMed2004

Polymorphism within the bovine estrogen receptor-alpha gene 5'-region.

Due to the functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered candidates for the markers of production and functional traits in farm animals, including cattle. In the present study, on the basis of the sequences of the human, ovine, and porcine ER genes, available in the GenBank database, sets of PCR primers were designed and used to amplify the bovine ERalpha gene 5'-region. Seven overlapping fragments of the 5' region of the bovine ERalpha gene were amplified and then sequenced. Altogether, these fragments were composed in the 2853-bp sequence which was deposited in the GenBank database under accession no. AY340597. The sequenced fragment included the noncoding exons A, B, C, their putative promoters, and a part of the coding exon 1. A polymorphism within the 5' region of the bovine ERalpha gene-A/G transition, which could be recognized with RFLP-BglI, lying upstream to the exon C, was identified for the first time using this sequence.

5' Flanking Region↗
PubMed2006

[Molecular characterization of full-length genome of Japanese encephalitis virus (02-76) newly isolated in China.].

BACKGROUND: To sequence and analyze the complete nucleotide sequence of the Japanese encephalitis virus (JEV) strain 02-76, newly isolated in 2002 in China and to provide information for the genomic structure of JEV and the characteristics of virulence. METHODS: Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments, sequencing was performed and all the nucleotides were connected to acquire the full-length genome. Computer software was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees including Clustal X(1.8), DNASTAR, GENEDOC(3.2). RESULTS: The result of sequence analysis showed that the genome of 02-76 strain was 10,977 nucleotides long. An open reading frame from 95 to 10,391 including 10,296 bases was found capable of coding for a 3432 amino acid polyprotein. Compared with the Beijing 1 strains isolated in 1949 in China, there was a 248 nucleotide divergence and 16 amino acid divergence. Comparison of the complete genome sequences of different JEV isolates showed a 0.6%-15.1% nucleotide sequence divergence among them, which resulted in 0.2%-4.6% amino acid sequence divergence. Phylogenetic analysis through PrM/C,E,3'NTR and full-length genome showed that the 02-76 strain belonged to genotype 3. CONCLUSION: Analysis based on the complete genome sequences of different JEV isolates showed that the 02-76 isolate in 2002 belonged to genotype 3 and was close to the old Chinese isolates SA-14.

China↗