Isolation and characterization of two replication-dependent mouse H1 histone genes.

Mice contain at least seven nonallelic forms of the H1 histones, including the somatic variants H1a-e and less closely related variants H1 degrees and H1t. The mouse H1 degrees and H1c (H1var.1) genes were isolated and characterized previously. We have now isolated, sequenced and studied the expression properties of two additional mouse H1 genes, termed H1var.2 and H1var.3. Extensive amino acid and nucleotide sequence comparisons were made between the two genes and other mammalian H1 histone genes. A high degree of nucleotide sequence identity was seen between the H1var.2, rat H1d and human H1b genes, even well beyond the coding region, indicating that these genes are likely homologues. Unlike the previously characterized mouse H1var.1 gene which produces both nonpolyadenylated and polyadenylated mRNAs, the H1var.2 and H1var.3 genes produce only typical, replication dependent, nonpolyadenylated mRNAs.     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996     

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome. Accession numbers AF015784 and AF015785 at the GenBank.     

Characterization of the beta2-microglobulin gene of the horse

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.     

Genomic structure, polymorphism and expression of ACCN1 and ACCN3 genes in the horse

A category of cation gate proteins was shown to be present in sensory neurons and act as receptors of protons present in tissues such as muscles. The Amiloride-sensitive Cation Channel, Neuronal (ACCN) gene family is known to play a role in the transmission of pain through specialized pH sensitive neurons. Muscles from horses submitted to strenuous exercises produce lactic acid, which may induce variable pain through ACCN differential properties. The sequences of the equine cDNAs were determined to be 2.6 kb in length with an open reading frame of 1539 bp for ACCN1 and 2.1 kb in length with an open reading frame of 1602 bp for ACCN3. The ACCN1 gene is 990 kb long and contains 10 exons, and the ACCN3 gene is 4.2 kb long and contains 11 exons. The equine ACCN1 and ACCN3 genes have an ubiquitous expression but ACCN1 is more highly expressed in the spinal cord. We identified one alternative ACCN3 splicing variant present in various equine tissues. These mRNA variants may encode two different protein isoforms 533 and 509 amino acids long. Ten single nucleotide polymorphisms (SNPs) were detected for ACCN1; five in the coding and five in the non-coding region, with no amino acid change, while the three SNPs identified in the coding region of the ACCN3 gene introduce amino acid changes. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species, especially for the ACCN1 gene.     

Nucleotide sequence of the B1-hordein gene of barley Hordeum vulgare L

The gene encoding B1 hordein of Hordeum vulgare (cv. Donetsky 4) was cloned and entirely sequenced. It contains no introns and codes for of 293 amino acids long polypeptide with molecular weight 33418. Our clone differs from the previously sequenced B1 hordein genes in some positions within the coding region (there are 4 nucleotide changes and a 12 bp deletion, as compared with the pB11 cDNA clone, and 5 nucleotide changes, as compared with the pBHP184 genomic clone). These changes result in polymorphism of amino acid sequences at 5 positions. 5'-flanking region contains putative regulatory and promoter sequences and differs from that of the pBHP184 clone in 3 positions.     

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.     

Bovine and feline gastrin cDNA sequences and the amino acid and nucleotide sequence homologies among mammalian species.

The complete nucleotide sequences of cDNAs encoding bovine and feline preprogastrins have been cloned from the antral mucosa mRNA. The gastrin mRNA of each animal encodes a preprogastrin of 104 amino acids consisting of a signal peptide, a prosegment of 37 amino acids, and a gastrin 34 sequence, followed by a glycine (the amide donor). The cleavage following a pair of lysine residues yields gastrin 17. We found that pairs of arginine residues flanking gastrin 34, the typical processing site sequence of all other preprogastrins and many peptide hormones, were arginines in the bovine preprogastrin, but the first basic amino acid pair had changed to Arg-Trp (57-58 residues) instead of Arg-Arg in the feline preprogastrin. Comparison of these amino acid and nucleotide sequences with published mammalian sequences showed extensive homology in the coding (63 to 73% amino acid identity) and in the untranslated regions (67 to 89% identity). Prosequence, the most variable region, shows greater amino acid difference between bovine and human preprogastrin (54% identity), and between bovine and rat preprogastrin (54% identity) than between other species (62 to 82% identity).     

Structure of the constant and 3' untranslated regions of the murine Balb/c gamma 2a heavy chain messenger RNA.

The complete sequence for the constant and 3' untranslated regions of a mouse gamma 2a immunoglobulin heavy chain mRNA is reported. The sequence is 1093 nucleotides long coding for the CH1 (amino-acids 118-214), the Hinge (215-230), the CH2 (231-340) and the CH3 (341-447). The 3' untranslated region is 103 nucleotides long preceding the poly(A). The nucleotide sequence predicts as in the case for gamma 1 and gamma 2b heavy chains an additional lysine residue before the termination codon. This sequence has been compared to the corresponding sequences of gamma 1 and gamma 2b heavy chain mRNAs. These sequences are respectively 75% and 84% homologous. The CH2 domains of gamma 2a and gamma 2b are 95% homologous at the nucleotide level. The cross-over point of a gamma 2a - gamma 2b heavy chain variant is located in a segment of 73 perfectly matching nucleotides. The 3' non coding regions of gamma 2a and gamma 2b are 89% homologous.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70

Background

The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages.

Methods

The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage.

Results

The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1.

Conclusions

These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest an independent evolutionary history for each type.