Putative Polyadenylation Signal Research Articles

The expressions of nr0b1/2 genes in the Chinese giant salamander and their response to estradiol benzoate/17α-methyltestosterone treatment

Previously, the nr0b1 and nr0b2 genes have been identified in various animals and found to be closely associated with sex determination. In order to investigate the regulatory roles of nr0b1 and nr0b2 genes in the sex determination of Chinese giant salamander (CGS, Andrias davidianus), we obtained the full-length cDNA sequences of nr0b1 and nr0b2 through cloning, determined the characteristics of their tissue expressions and the responses of nr0b1 and nr0b2 to estradiol benzoate (EB) and 17α-methyltestosterone (MT) by using quantitative real-time PCR (qRT-PCR), and evaluated gonad development through histological sections. The full-lengths of cDNA of nr0b1/2 in C GS are separately 1278bp and 789bp. They have complete open reading frames (ORF) of 864bp and 774bp and encode 287 and 257 amino acids respectively. The 3' untranslated regions (UTR) are 276bp and 15bp, and the 5' UTR of nr0b1 is 138bp. In the 3' UTR of nr0b1, a putative polyadenylation signal (AATAAA) is identified. Tissue distribution analysis indicates that nr0b1 is mainly expressed in the pituitary, kidney, and testes, with significant differences between females (ZW-F) and males (ZZ-M) expression in the testes. On the other hand, nr0b2 is mainly expressed in the liver, followed by gonadal tissues. After immersion and feeding with EB and MT, EB's promotion on nr0b1 expression in CGS ovaries and testes is shown, by contrast MT only has a positive effect on nr0b2 in CGS testes. Histological sections show that after EB treatment, both genetic females (ZW-F) and sex-reversed females (ZZ-F) of CGS contain oogonia, indicating that EB can cause sex reversal in CGS; however, after MT treatment, no cellular morphological changes are observed either in genetic females (ZW-F) or in genetic males (ZW-M), indicating that this concentration cannot induce a shift in the sex ratio of CGS. These findings lay the foundation for further investigating the molecular mechanisms by which nr0b1/2 influence the differentiation and maintenance of CGS gonads.

Iron restriction increases the expression of a cytotoxic cysteine proteinase TvCP2 by a novel mechanism of tvcp2 mRNA alternative polyadenylation in Trichomonas vaginalis

Trichomonas vaginalis TvCP2 (TVAG_057000) is a cytotoxic cysteine proteinase (CP) expressed under iron-limited conditions. This work aimed to identify one of the mechanisms of tvcp2 gene expression regulation by iron at the posttranscriptional level. We checked tvcp2 mRNA stability under both iron-restricted (IR) and high iron (HI) conditions in the presence of actinomycin D. Greater stability of the tvcp2 mRNA under the IR than in HI conditions was observed, as expected. In silico analysis of the 3′ regulatory region showed the presence of two putative polyadenylation signals in the tvcp2 transcript. By 3′-RACE assays, we demonstrated the existence of two isoforms of the tvcp2 mRNA with different 3′-UTR that resulted in more TvCP2 protein under IR than in HI conditions detected by WB assays. Additionally, we searched for homologs of the trichomonad polyadenylation machinery by an in silico analysis in the genome database, TrichDB. 16 genes that encode proteins that could be part of the trichomonad polyadenylation machinery were found. qRT-PCR assays showed that most of these genes were positively regulated by iron. Thus, our results show the presence of alternative polyadenylation as a novel iron posttranscriptional regulatory mechanism in T. vaginalis for the tvcp2 gene expression.

High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis.

Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.

Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals.

The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3′-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3′ end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression. Research on TTs is not limited to transgene expression but could be extended to the introduction of appropriate mutations into TTs via genome editing, paving the way for expression modulation of endogenous genes.

Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene.

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.

Molecular characterization of polymeric immunoglobulin receptor and expression response to Aeromonas hydrophila challenge in Carassius auratus

The polymeric immunoglobulin receptor (pIgR) plays a pivotal role in mucosal immune response by transporting polymeric immunoglobulins onto the surface of mucosal epithelia to protect animals from invading pathogens. In this study, the full-length cDNA of pIgR was firstly cloned in Qihe crucian carp (Carassius auratus), hereafter designated as CapIgR, by using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The molecular characterization and expression of CapIgR were investigated. The full-length cDNA sequence of CapIgR was composed of 1409 bp, which included a 112 bp 5ʹ-untranslated region (UTR), a 984 bp ORF, and a 313 bp 3ʹ-UTR, with a putative polyadenylation signal sequence AATAAA located upstream of the poly(A) tail. The deduced amino acid sequence indicated that CapIgR was a single-spanning transmembrane protein with 327 amino acids and possessed a signal peptide, an extracellular region containing two immunoglobulin-like domains, a transmembrane region, and an intracellular region. The mRNA expression levels of CapIgR were detected in different tissues of healthy C. auratus by quantitative real-time PCR, and the highest expression level was found in the liver. After Aeromonas hydrophila challenge, CapIgR expression was upregulated in different tissues at certain time points, and temporal expression changes of CapIgR fluctuated in a time-dependent manner. CapIgR exhibited rapid immune response to A. hydrophila challenge and played an important role in the immune defense of fish. These findings provided insights into the structure, function, and immune defense mechanism of CapIgR in C. auratus. This study can serve as a basis for developing disease control strategies in aquaculture.

Deep RNA Sequencing Reveals Hidden Features and Dynamics of Early Gene Transcription in Paramecium bursaria Chlorella Virus 1

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

Competition within Introns: Splicing Wins over Polyadenylation via a General Mechanism

Most eukaryotic messenger RNAs are capped, spliced, and polyadenylated via co-transcriptional processes that are coupled to each other and to the transcription machinery. Coordination of these processes ensures correct RNA maturation and provides for the diversity of the transcribed isoforms. Thus, RNA processing is a chain of events in which the completion of one event is coupled to the initiation of the next one. In this context, the relationship between splicing and polyadenylation is an important aspect of gene regulation. We have found that cryptic polyadenylation signals are widely distributed over the intron sequences of Drosophila melanogaster. As shown by analyzing the distribution of genes arranged in a nested pattern, where one gene is fully located within an intron of another gene, overlapping of putative polyadenylation signals is a fairly common event affecting about 17% of all genes. Here we show that polyadenylation signals are silenced within introns: the poly(A) signal is utilized in the exonic but not in the intronic regions of the transcript. The transcription does not end within the introns, either in a transient reporter system or in the genomic context, while deletion of the 5'-splice site restores their functionality. According to a full Drosophila transcriptome analysis, utilization of intronic polyadenylation signals occurs very rarely and such events are likely to be inducible. These results confirm that the transcription apparatus ignores premature polyadenylation signals for as long as they are intronic.

Genome-Wide Control of Polyadenylation Site Choice by CPSF30 in Arabidopsis

The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) has been implicated in the responses of plants to oxidative stress, suggesting a role for alternative polyadenylation. To better understand this, poly(A) site choice was studied in a mutant (oxt6) deficient in CPSF30 expression using a genome-scale approach. The results indicate that poly(A) site choice in a large majority of Arabidopsis genes is altered in the oxt6 mutant. A number of poly(A) sites were identified that are seen only in the wild type or oxt6 mutant. Interestingly, putative polyadenylation signals associated with sites that are seen only in the oxt6 mutant are decidedly different from the canonical plant polyadenylation signal, lacking the characteristic A-rich near-upstream element (where AAUAAA can be found); this suggests that CPSF30 functions in the handling of the near-upstream element. The sets of genes that possess sites seen only in the wild type or mutant were enriched for those involved in stress and defense responses, a result consistent with the properties of the oxt6 mutant. Taken together, these studies provide new insights into the mechanisms and consequences of CPSF30-mediated alternative polyadenylation.

140 CHARACTERIZATION OF THE BOVINE ANTISENSE TO INSULIN-LIKE GROWTH FACTOR TYPE 2 RECEPTOR (bAIRN) NONCODING RNA

Bovine insulin-like growth factor type 2 receptor (IGF2R) is an imprinted gene whose aberrant expression has been implicated in development of abnormal offspring syndrome. Gene IGF2R is a member of the IGF2R/AIRN imprinted gene cluster located on chromosome 9 in cattle. This imprinting cluster is flanked on the proximal 5′ end by MAS1. In the mouse, the antisense to Igf2r noncoding RNA, Airn, is a 108-kb polyadenylated transcript that regulates imprinted expression of Igf2r and other genes within the Igf2r/Airn cluster in association with implantation. Bovine AIRN (bAIRN) is expressed in post-implantation fetal tissues with expression coincident with imprinted expression of IGF2R. Further characterisation of bAIRN is needed before investigation of its potential role in regulating imprinted expression of IGF2R in cattle can be pursued. Therefore, the objective of this study was to identify the length and key characteristics of the bAIRN ncRNA transcript. The PCR primer sets (n = 26) were designed based on genomic sequences to walk down the predicted bAIRN ncRNA sequence by amplifying key segments located along the transcript. Total RNA, extracted from gestational day 150 bovine fetal liver, was DNase-treated to eliminate contaminating genomic DNA. The DNase-treated RNA was then used to generate cDNA as a template for PCR amplification. A putative promoter region containing the transcriptional start site for bAIRN was identified based on NCBI sequence data. This bAIRN promoter is located 441 bp upstream of differentially methylated region 2 (DMR2) within intron 2 of IGF2R and contains a CAAT-box, GC-box and TATA-box. The PCR primer sets (n = 2) designed to amplify products upstream of the putative promoter failed to produce any PCR amplicons. However, PCR primer sets (n = 6) designed to amplify segments located from 4 to 10 kb downstream of the putative promoter region produced amplicons that were sequence verified to be bAIRN ncRNA. Additional PCR primer sets (n = 3) produced sequence-verified amplicons that were located ∼27, 45 and 52 kb downstream of the putative bAIRN transcription start site. Between 27 and 44 kb downstream of the bAIRN promoter, a series of sequence duplications from MAS1 were identified within the bAIRN ncRNA. The role of these duplicated regions is currently unknown. Three PCR primer sets (n = 3) designed to amplify consecutive regions spanning from 93.4 to 101 kb downstream of the bAIRN promoter failed to produce amplicons, suggesting prior termination of the bAIRN transcript. In addition, a strong putative polyadenylation signal was identified at 65.4 kb downstream from the bAIRN promoter. In summary, the full-length bAIRN ncRNA has been characterised. The bAIRN promoter is located ∼440 bp upstream of DMR2 on the IGF2R antisense strand. Transcription from the bAIRN promoter results in a noncoding RNA with a length of ∼65 kb. The central third of the bAIRN transcript contains a series of sequence duplications from MAS1. The function of these duplications within the bAIRN ncRNA is currently unknown. This research was supported by the North Carolina Agricultural Experiment Station.

Dietary sodium alginate administration enhances Mx gene expression of the tiger grouper, Epinephelus fuscoguttatus receiving poly I:C

The full-length complementary (c)DNA of the Mx gene was cloned from the spleen of the tiger grouper, Epinephelus fuscoguttatus, injected with poly I:C by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The 2163-bp cDNA contained an open reading frame (ORF) of 1878 bp, a 42-bp 5′-untranslated region (UTR), and a 243-bp 3′-UTR including a stop codon (TAG), a poly A tail, and a putative polyadenylation signal (AATAAA) 14 bp upstream of the poly A tail. The ORF encoded a protein of 626 amino acids (aa) with a predicted molecular mass of 71.3 kDa and an estimated p I of 5.55. The grouper Mx sequence contained putative functional domains including a tripartite guanosine-5′-triphosphate (GTP)-binding motif (GDQSSGKS, DLPG, and TKPD), the signature of the dynamin family (LPRGSGIVTR), and a putative leucine zipper in the carboxyl-terminal end of the protein. Sequence comparison showed that the Mx gene of this grouper had overall respective identities of 70%–96%, 51%–56%, and 34% to those of teleosts, mammals, and the abalone ( Haliotis discus discus), respectively. A phylogenetic analysis revealed that the cloned Mx (TGMx) from tiger grouper was more-correlated with orange-spotted grouper MxII (gMxII). TGMx was constitutively expressed in all tissues, and its expression was upregulated by poly I:C and lipopolysaccharide (LPS), especially after a poly I:C injection. The Mx expressions had significantly increased in the eyes, head kidney, spleen, intestines, liver and gills after the LPS injection, and in all tested tissues after the poly I:C injection as compared to the groupers injected with PBS. The quantitative real-time RT-PCR analysis showed that Mx mRNA expression in the spleen and head kidney of grouper fed a diet containing sodium alginate and then injected with poly I:C after 12 h significantly increased, compared to those of fish fed a control diet (without sodium alginate). We therefore recommend an LPS and poly I:C injection to induce TGMx expression which was further enhanced by dietary sodium alginate administration.

Research note: Analysis of expressed sequence tags from the chrysophycean alga Ochromonas danica (Heterokontophyta)

SUMMARY A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′-end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E-value <10−4) to sequences in a public database. The annotation of these ESTs was carried out to assess subcellular localization of the putative proteins using several internet-accessible prediction programs for subcellular localization. These analyses revealed that putative plastid proteins in Ochromonas possess N-terminal bipartite presequences with a conserved phenylalanine at the N-terminus of the predicted transit peptide-like domains, similar to other ‘red-lineage’ secondary symbiotic organisms. The examination of sequences of 3′-UTR revealed that, similarly to chlorophyte algae, UGUAA may represent a putative polyadenylation signal in O. danica.

Isolation and Expression of a Novel Alligator Gene Belonging to the Sox Gene Family

Sox genes share a highly conserved DNA-binding motif, the HMG (high mobility group)-box domain, and have diverse roles in vertebrate embryonic development. A novel SRY-related cDNA (temporarily called Sox33) isolated from the Chinese alligator (Alligator sinensis) is 1,819 bp in length, with an open reading frame from 220 to 1113 bp, encoding a protein of 298 amino acids. Two putative polyadenylation signal sequences (AATAAA) are present upstream of the poly(A) tail in the 3' UTR (at 1255-1260 and 1774-1779). The putative protein contains an HMG-box domain most closely related to hSox12, mSox4, rtSox11, and mSox11 homologs, indicating that alligator Sox33 belongs to group C in the Sox gene family. Alligator adult and developing tissues were tested for Sox33 mRNA by independent Northern blots using a 336-bp probe (at 907-1243) between the HMG-box and the poly(A) site I and a 277-bp probe (at 1477-1754) between the two polyadenylation sites. Two transcripts (1.3 kb and 1.8 kb) in developing brain and one (1.8 kb) in adult brain were identified by the 336-bp probe; only one transcript (1.8 kb) in developing and adult brains was detected by the 277-bp probe. The results suggest that alligator Sox33 may use a different polyadenylation mechanism in the developing brain and play a role in the development and maintenance of the nervous system.

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Analyses of the cDNA and genomic DNA sequences encoding the luteinizing hormone β-subunit precursor protein in the rabbit

To examine the molecular basis for efficient induction of superovulation in the rabbit, we determined the cDNA sequences of the luteinizing hormone β-subunit (LHB) from Japanese White (JW), New Zealand White (NZW), and Dutch-Belted (Dutch) rabbits, and we compared these LHB sequences with those of other mammals. Using 5′- and 3′-rapid amplification of cDNA ends (RACE) with pituitary cDNA libraries, we found that the LHB cDNAs of all three breeds are the same length (523 bp from the 5′-end to the polyA site) and have putative AATAAA polyadenylation signal sequences at nucleotides 504 to 509. Northern blot analysis indicated that the ∼600-nt mRNA encoding JW LHB is slightly longer than the LHB mRNAs of the other two breeds. The NZW and Dutch rabbit LHB coding sequences are 426 bp long, and their G + C contents are higher (>73%) than those of other mammalian LHBs (<70%). The predicted 141-amino-acid sequences of the JW and NZW LHB proteins are identical, and the Dutch LHB and JW/NZW sequences differ at only two residues. The exon–intron configuration of the NZW LHB gene (three exons and two introns) is similar to that of other mammalian LHB genes, and the sequences of NZW rabbit and other mammalian LHB promoter regions are highly conserved. Phylogenetic analysis of the deduced amino acid sequences of the three rabbit LHB proteins indicated that the rabbit occupies a phylogenetic position between rodents and domestic animals, and is far from humans. The results suggest that LH prepared from rodents or domestic animals, if available, would be a better inducer for superovulation in rabbits than human LH/CG.

Characterization of a new densovirus infecting the German cockroach, Blattella germanica

A new DNA virus (Parvoviridae: Densovirinae, Densovirus) was isolated and purified from descendants of field-collected German cockroaches, Blattella germanica. Viral DNA and cockroach tissues infected with B. germanica densovirus (BgDNV) were examined by electron microscopy. Virus particles, about 20 nm in diameter, were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule of about 1.2 microm in length. BgDNV isolated from infected cockroaches infected successfully and could be maintained in BGE-2, a B. germanica cell line. The complete BgDNV genome was sequenced and analysed. Five open reading frames (ORFs) were detected in the 5335 nt sequence: two ORFS that were on one DNA strand encoded structural capsid proteins (69.7 and 24.8 kDa) and three ORFs that were on the other strand encoded non-structural proteins (60.2, 30.3 and 25.9 kDa). Three putative promoters and polyadenylation signals were identified. Structural analysis of the inverted terminal repeats revealed the presence of extended palindromes. The genome structure of BgDNV was compared with that of other members of the family Parvoviridae; the predicted amino acid sequences were aligned and subjected to phylogenetic analyses.

Sequence analysis of mRNA polyadenylation signals of rice genes

The formation of eukaryotic mRNAs involves the cleavage and polyadenylation of pre- mRNAs. To investigate the sequence requirement of putative polyadenylation signals (PASs), poly(A) sites and downstream elements (DUEs) in 3′-end-pro- cessing in rice, we compared expressed sequences tags (ESTs) with poly(A) extremity to full-length cDNA sequences and constructed a database of 12969 pre- mRNA sequences in ?40―+40 nt surrounding the poly(A) sites, which were from 9953 genes. The al- ternative poly(A) sites were revealed in approxi- mately 25% of mRNAs. Nearly 80% of pre-mRNAs showed stringent requirement of the YA (CA or UA) at poly (A) sites for polyadenylation. About 7.9% had the AAUAAA signals on ?40―?1 nt upstream of the poly(A) sites. Over 60% of mRNAs probably used the one- or two-base variants of AAUAAA hexamers as their PASs in 3′ fragments. The single-base variants of AAUGAA revealed the high frequency in 11.5% of 3′ fragments. The DUEs were detected in 90% of pre- mRNAs, especially more than half of the pre-mRNAs with multi-base variants of AAUAAA had the DUEs surrounding the poly(A) site. The location of DUE is also important for defining the cleavage site. Al- though most of the rice pre-mRNAs did not contain AAUAAA signal, the existence of downstream ele- ments ensured the efficiency of cleavage-polyade- nylation

Sequence analysis of cDNA encoding rabbit follicle-stimulating hormone β-subunit precursor protein

To understand the molecular basis of the rabbit’s efficient superovulation, we determined the cDNA sequence of the follicle-stimulating hormone (FSH) β-subunit precursor protein using a combination of 5′- and 3′-rapid amplification of cDNA ends (RACE) with pituitary cDNA libraries of the Japanese White rabbit and compared it with those of other mammals. RACE experiments detected at least three transcripts for the FSHβ precursor protein in the libraries. The transcripts had lengths of 457, 1621, and 1767 bp, from the 5′-end to the poly(A) site. The shortest and mid-length transcripts had the putative polyadenylation signal sequence AATAAA at nucleotides 436 and 1601, respectively, whereas the longest form had an ATTAAA sequence at nucleotide 1745 of the cDNA sequence. These transcripts are likely to be polyadenylation variants of one large transcript because they share the same coding sequence for the precursor protein (130 amino acid residues in length). However, only a few shortest variants seem to be formed because the shortest variants were not detected by Northern blot analysis. Phylogenetic analysis of the deduced amino acid sequence indicates that the rabbit is phylogenetically closer to humans than to the other mammals, suggesting that an FSH preparation from human sources would be superior as a follicle stimulant for the induction of superovulation.

Gonadotroph-specific expression of the human follicle stimulating hormone β gene in transgenic mice

A paucity of in vitro models has hampered studies of molecular mechanisms of FSH subunit gene expression. Consequently, we used an in vivo transgenic strategy to map the location of regulatory elements in the cloned 10 kb human FSHβ gene. Analyses of transgenic mouse lines revealed that successive 5′ truncations of the hFSHβ promoter region to −350 bp relative to the transcriptional initiation site retained gonadotroph-specific expression and the sexually dimorphic pattern of male greater than female FSHβ mRNA levels found normally in rodent pituitary. Truncation of the 3′ flanking sequences from positions +3142 to +2138 bp relative to the translational stop codon in exon 3 resulted in a complete loss of transgene expression, suggesting the presence of critical regulatory elements mapping to the 1 kb genomic segment downstream of position +2138, in addition to the proximal 5′ promoter elements. In silico phylogenetic comparisons of mammalian FSHβ genes revealed five islands of highly conserved sequence homology corresponding precisely to the proximal 5′ promoter region, exon 2, the 5′ translated region of exon 3, and two regions at the 3′ untranslated end of exon 3 that include putative polyadenylation and transcriptional termination signals. Sequence analyses of the 5′ proximal promoter revealed the presence of several putative homeodomain binding sites as well as GATA, SMAD, AP-1, NF-1, NF-Y and steroid hormone transcription factor binding sites within the highly conserved −350 bp promoter region. Notably absent from these 5′ sequences, however, are consensus binding sites for either Egr-1 or Lim-2 transcription factors known to be critical for the gonadotroph-specific expression of the LHβ gene. These findings support the hypothesis that one of the mechanisms underlying the differential regulation of the LHβ, FSHβ, and common α-gonadotropin subunits within pituitary gonadotrophs may be differences in sequence-specific binding requirements for distinct combinations of transcription factors.

The AAUAAA Motif of Bamboo Mosaic Virus RNA Is Involved in Minus-Strand RNA Synthesis and Plus-Strand RNA Polyadenylation

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5'-cap structure and a 3' poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3' untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3' UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.