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Isolation and characterization of genomic and transcribed retrotransposon sequences from sorghum.

Reverse transcriptase sequences from both major classes of retrotransposons were amplified from sorghum genomic DNA, leaf mRNA and callus protoplast mRNA. Sequence analysis of clones derived from genomic DNA demonstrated the presence of a wide variety of copia-like and gypsy-like elements. Twenty-four families of copia-like elements were found, of which at least thirteen were expressed in callus protoplasts. Two families (containing forty-eight subfamilies) of gypsy-like elements were discovered, both closely related to Huck of maize. At least twenty-seven of these subfamilies were expressed in callus protoplasts. Most of these elements were expressed at high levels in protoplasts derived from embryogenic callus, but expression of only a few was detected (at low levels) in leaves. Sequence divergence within individual families was quite high, and all relatedness profiles were consistent with vertical transmission of these elements. These data indicate that sorghum contains a large number and diversity of retrotransposons, and that some may be useful as transposon tagging systems in callus protoplasts.

Base Sequence↗

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

Animals↗

Karyotypic characterization and genomic organization of the 5S rDNA in Erpetoichthys calabaricus (Osteichthyes, Polypteridae).

Polypterids are a group of Osteichthyan fish whose evolutionary relationships with closer basal ray-finned and lobe-finned fish have been disputed since their discovery. Very little is known about the evolutive karyology in the whole Polypteriformes group. In order to fill this gap, a cytogenetic analysis of Erpetoichthys calabaricus species was performed, using both classical and molecular techniques. Karyotype structure (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG)n and ribosomal 5S and 18S rRNA genes were examined in twenty specimens of E. calabaricus by using Ag-NOR, classical C-banding, sequential CMA3/4',6-diaminidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH). CMA3 marked all centromerical and some (no. 1 and no. 15) telomeric regions. Staining with Ag-NOR and CMA3 showed the presence of two NORs on the p arm of the chromosome pair no. 1. Hybridization with telomeric probes (TTAGGG)n showed signals at the end of all chromosomes. 5S rDNA was cloned and sequenced. After the alignment, the 5S rRNA sequences revealed an organization made up of two different classes of tandem arrays (type I and type II). FISH with 5S rDNA marked the telomeric regions of the small chromosome pair no. 15, while FISH with 18S rDNA marked the telomeric region of the pair no. 1. The results obtained were compared with cariological data on closer species now available in literature.

Animals↗

Expression of the major inner capsid protein of the group B rotavirus ADRV: primary characterization of genome segment 5.

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.

Amino Acid Sequence↗

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.

Amino Acid Sequence↗

Molecular cloning, characterization, and genomic localization of a human potassium channel gene.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of resting membrane potential, and development of memory and learning. In addition to their role in regulating myocyte excitability, cardiac K+ channels control heart rate and coronary vascular tone and are implicated in the development of arrhythmias. We report here the cloning and sequencing of a k+ channel gene, KCNA1, derived from a human cardiac cDNA library and the chromosomal localization of the corresponding genomic clone. Oligonucleotides based on a delayed rectifier K+ channel gene were used in PCR reactions with human genomic DNA to amplify the S4-S6 regions of several different K+ channel genes. These sequences were used to isolate clones from a human cardiac cDNA library. We sequenced one of these clones, HCK1. HCK1 contains putative S2-S6 domains and shares approximately 70% sequence homology with previously isolated Shaker homologues. HCK1 was used to screen human cosmid libraries and a genomic clone was isolated. By sequencing the genomic clones, a putative S1 domain and translation initiation sequences were identified. Genomic mapping using human-rodent somatic cell panels and in situ hybridization with human metaphase chromosomes have localized KCNA1 to the distal short arm of human chromosome 12. This work is an important step in the study of human cardiac K+ channel structure and function and will be of use in the study of human inherited disease.

Amino Acid Sequence↗

Characterization of genomic instability in ulcerative colitis neoplasia leads to discovery of putative tumor suppressor regions.

Ulcerative colitis (UC) is an inflammatory disease of the colon that is associated with increased risk of colorectal cancer associated with genomic instability. We have previously demonstrated that genomic instability is present in UC patients with colonic neoplasia, and hypothesized that the chromosomal alterations may be taking place in regions that are susceptible to mutation or that provide a growth advantage to a cell undergoing neoplastic transformation. In this study, we used two polymerase chain reaction (PCR)-based DNA fingerprinting techniques (arbitrarily primed PCR and inter-simple-sequence-repeat PCR) to study the process of genomic instability. The two techniques of DNA fingerprinting cross-validate the instability observed in these studies. We analyzed the molecular basis of 10 commonly altered DNA bands obtained from DNA fingerprints of biopsies from various histologic grades of UC patients with dysplasia or cancer (UC Progressors). We determined that the band changes in the fingerprint truly represent changes in DNA sequence, and that the fingerprinting provides highly reproducible results. Furthermore, our investigation revealed that 40% of alterations involve repetitive sequences. Two frequently deleted sequences in 6q27 and 2q14 were studied further because they were frequently abnormal in the dysplastic and nondysplastic tissue of UC Progressors. The losses from 6q27 and 2q14 were confirmed by loss of heterozygosity and real-time PCR analysis. Both of these regions in chromosomes 6 and 2 are surrounded by highly repetitive and mobile LINE-1 elements, possibly making the region susceptible to mutational change. These regions were affected (lost) in UC Progressors but not in UC patients who were neoplasia free. Loss of heterozygosity at 6q27 has been described in ovarian and other cancers, while the 2q14 region has been implicated in prostate and sporadic colon cancers. Both regions are likely to contain tumor-suppressor genes. In conclusion, the genomic instability in UC Progressors can occur in regions that are susceptible to change and are locations of putative tumor-suppressor genes.

Colitis, Ulcerative↗

Rhodococcus dendrobeaniae sp. nov., an actinomycete isolated from an Arctic marine invertebrate exhibiting cytotoxic activity, and an emended description of Rhodococcus sovatensis.

A polyphasic study was conducted to establish the taxonomic status of strain T060T, an orange, aerobic, coccoid and non-motile actinomycete, isolated from a marine bryozoan (Dendrobeania sp.) collected in the Barents Sea. Phylogenetic analysis of the 16S rRNA gene sequences revealed Rhodococcus sovatensis DSM 102881T as the closest related species to strain T060T with a similarity of 99.54%. Phylogenomic analysis confirmed a close relationship between T060T and R. sovatensis DSM 102881T, while supporting their distinction. Digital DNA-DNA hybridization and average nucleotide identity values between strain T060T and R. sovatensis DSM 102881T were 26.4 and 84.3%, respectively, supporting the delineation of the isolate as a new species. Genomic characterization of the assembled genomes of T060T and R. sovatensis DSM 102881T showed genome sizes of 5.3 and 4.2 Mbp, with a G + C content of 64.38 and 65.01%, respectively. Genome analysis of strain T060T identified 15 biosynthetic gene clusters (BGCs) with low sequence similarity to known BGCs, indicating its capacity to produce unknown, potentially bioactive secondary metabolites. Furthermore, growth of T060T in eight different media revealed condition-dependent cytotoxic activity. The strongest cytotoxicity was observed for fractionated extracts from T060T grown in half-strength ISP 2 with filtered seawater against the human malignant cell lines MCF7 and A2058, and to a lesser extent against the non-malignant MRC5 cell line, highlighting its biotechnological potential. Based on the data from polyphasic taxonomy studies, it is proposed that strain T060T be classified in the genus Rhodococcus as Rhodococcus dendrobeaniae sp. nov. Additionally, the acquired data of R. sovatensis were used to amend its original description.

Rhodococcus↗

Genome-wide characterization of the FOX gene family reveals sex-biased expression and FoxO-associated regulation during gonadal development in Bellamya aeruginosa.

Forkhead box (FOX) transcription factors are important regulators of development and reproduction, but their roles in molluscan gonadal development remain unclear. In this study, the FOX gene family was systematically identified in the freshwater gastropod Bellamya aeruginosa, and comparative gonadal transcriptomic analyses were performed across sexes and developmental stages.A total of 35 FOX genes were identified and classified into 19 subfamilies. Phylogenetic and synteny analyses indicated that the FOX gene family is generally conserved among mollusks, whereas FoxG and FoxL2 showed lineage-specific expansion. Structural analyses demonstrated that most BaeFOX proteins retained the conserved Forkhead domain architecture. Transcriptomic analyses revealed that gene expression divergence between testes and ovaries was markedly greater than that between developmental stages within the same gonad, indicating that sexual dimorphism is the major source of gonadal transcriptional variation. Functional enrichment consistently identified the FoxO signaling pathway in sex-biased comparisons. Several FOX genes also displayed clear gonad-biased expression patterns, and network analyses suggested that BaeFoxO, BaeFoxL2, and BaeFoxG may play central regulatory roles in gonadal development. These findings provide the first comprehensive characterization of the FOX gene family in B. aeruginosa and suggest that FOX-related regulatory networks potentially participate in gonadal development in gastropod mollusks.

Animals↗

Characterization of genomic alterations in hepatoblastomas. A role for gains on chromosomes 8q and 20 as predictors of poor outcome.

As data on the genomic alterations in hepatoblastoma (HB) are limited, 34 HB tumors and three HB cell lines were screened for DNA copy number changes by comparative genomic hybridization. The average number of chromosomal imbalances per tumor was 2.3 +/- 0.5 (mean +/- SEM) with gains sevenfold more frequent than losses. The most frequent gains of chromosomal material in HB tumors were on 2q (44%), 1q (41%), 2p (29%), 20 (24%), 22q (18%), 8q (15%), 8p and 12q (9% each), as well as 7q, 12p, and 17 (6% each) and the only recurrent loss was on 4q in 12% of cases. Highly amplified sequences were identified in four tumors and mapped to 2q24 in two cases, to 8q in two cases (once to 8q11.2-q13 and once to 8q11.2-q21.3) as well as to 10q24-q26 in one case. In one cell line, highly amplified DNA sequences were mapped to 7p and 8q. Comparison to previously published data on this series of HB revealed that the number of chromosomal imbalances was significantly higher in HB tumors with loss of heterozygosity on 11p (P = 0.03), whereas in five of 10 HB biopsies without chromosomal imbalances, beta-catenin gene mutations were found. HB patients were divided into a good (no evidence of disease) and a poor (died of disease) outcome group according to their clinical course after standard therapy. Two alterations were found to be significantly associated with poor outcome: gain on 8q (P = 0.007) and gain on 20 (P = 0.009). In summary, our analysis allowed the identification of gains on chromosomes 1q and 2 as hallmark DNA copy number changes in HB with 2q24 as a critical chromosomal band. Furthermore, this study provided evidence that gains on 8q and 20 play a role as markers of prognostic significance in HB.

Adolescent↗

Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss).

Natural killer cell enhancement factor (NKEF) belongs to the antioxidant protein family. In the human, NKEF has the ability to enhance natural killer cell cytotoxic activity in vitro. In the present work, the cDNAs of NKEF from three strains of homozygous clones of rainbow trout were cloned from the splenic cDNA library of one of the strains, OSU142, and then by RT-PCR for the Hot Creek (HC) and Arlee (AR) strains. The HC sequence has 99% sequence identity with both OSU142 and AR. OSU142 and AR have only one nucleotide difference in the cDNA sequence. All three sequences have the same deduced NKEF peptide, which contains 199 amino acids. The 6. 5 kb genomic DNA of OSU142 containing NKEF was sequenced and contains six exons and five introns. Tissue specific expression of NKEF was studied by RT-PCR in eight different tissues of OSU142 and revealed that all tissues expressed NKEF. A southern blot revealed that the gene for NKEF is present in a single copy. The cDNA and amino acid sequences of trout NKEF have high similarity with human, rat, mouse and carp sequences, therefore, indicating that NKEF is a very conserved gene.

Amino Acid Sequence↗

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure.

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.

1-Methyl-3-isobutylxanthine↗

Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.

Human carbamyl phosphate synthetase I (CPSI) is an essential hepatic enzyme that initiates the urea cycle. Deficiency of this enzyme usually results in lethal hyperammonemia. CPSI is encoded by the CPSI gene located on chromosome 2q35. In the present study, we report the coding sequence and define the intron-exon structure of the human CPSI gene. These data are compared to the previously defined rat CPSI gene structure. This work was generated from direct sequence determination of human genomic DNA (35 introns) and comparison to public domain sequence of anonymous BACs (2 introns). The human CPSI gene spans >120kb of genomic DNA. CPSI has 38 exons and 37 introns, and all adhere to the consensus splicing sequences. Comparison of the human and rat CPSI genes reveals that the nucleotide sequences, amino acid sequences, and intron-exon organizations are highly similar. We report the primers and conditions for screening the human CPSI exonic and bordering intronic sequences. We also screened 100 individuals for polymorphisms in the human CPSI gene and identified 14 polymorphisms in the CPSI message. The knowledge of the CPSI gene structure and the 14 polymorphisms presented in this study will greatly facilitate future molecular studies involving the CPSI gene and the enzyme it encodes.

Animals↗

Characterization and genomic mapping of chimeric ERV9 endogenous retroviruses-host gene transcripts.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We performed Northern blot experiments using sequences from ERV9-LTR, and we observed a different pattern of expression in several different hemopoietic tumor cell lines. It is possible that by the result of a somatic integration event, or by virtue of their original dispersal in the genome, ERV9-LTRs may specifically induce the expression of different cellular sequences in different cell lineages. Here, we describe the identification and analysis of four chimeric cDNA clones isolated from the T-lymphoma Peer cell line, having a structure consistent with transcription initiation from an ERV9-LTR. All the cDNA clones represent transcripts derived from unique cellular sequences. We also report the genomic localization of these cDNA clones.

Base Sequence↗