Review

Transcription termination and 3′ processing: the end is in site!

Stem-loop binding protein (SLBP) binds a stem-loop structure of the mRNA, which is important for the stability of histone mRNAs and translation process. In the present study, two slbp cDNAs (Ecslbp1 and Ecslbp2) were cloned from a protogynous hermaphroditic orange-spotted grouper, Epinephelus coioides. Ecslbp1 cDNA contained a 678 base pair (bp) open reading frame (ORF), encoding a predicted polypeptide of 225 amino acids. Ecslbp2 cDNA contained a 1041 bp, encoding a predicted protein of 346 amino acids. The result of real-time PCR revealed that Ecslbp2 mRNA was exclusively detected in the ovary. Moreover, it was found to be restricted to oocytes according to in situ hybridization (ISH) analysis. Ecslbp2 was found to be hardly detected in gonia and significantly increase in the cytoplasm of primary-growth stage oocytes, but decreased during the process of vitellogenesis. Interestingly, Ecslbp2 expression centralized as a perinuclear speckle in early-primary-growth stage oocytes, which appeared to form into the Balbiani body (Bb) in late-primary-growth stage oocytes. These data indicated that Ecslbp2 might play an important role in the process of oocyte development, and could serve as an oocyte-specific molecular marker for the study of ovary development and sex reversal in groupers.

This chapter covers the broad field of transcription and epigenetic regulatory mechanisms to equip the gastrointestinal physiologist with the tools to understand gene expression. This chapter reviews the composition of the nucleic acids and the methods used to study their structure and function. The effect of noncoding RNA species such as microRNA and long noncoding RNAs is discussed. In addition, the impact of histone and DNA modification on gene expression collectively known as epigenetic influences are discussed in the context of how the gut microenvironment.

Previously, we observed that the transcripts of Invertebrate iridescent virus 6 (IIV6) are not polyadenylated, in line with the absence of canonical poly(A) motifs (AATAAA) downstream of the open reading frames (ORFs) in the genome. Here, we determined the 3′ ends of the transcripts of fifty-four IIV6 virion protein genes in infected Drosophila Schneider 2 (S2) cells. By using ligation-based amplification of cDNA ends (LACE) it was shown that the IIV6 mRNAs often ended with a CAUUA motif. In silico analysis showed that the 3′-untranslated regions of IIV6 genes have the ability to form hairpin structures (22–56 nt in length) and that for about half of all IIV6 genes these 3′ sequences contained complementary TAATG and CATTA motifs. We also show that a hairpin in the 3′ flanking region with conserved sequence motifs is a conserved feature in invertebrate-infecting iridoviruses (genus Iridovirus and Chloriridovirus).

Squamous cell carcinomas (SCCs) are the second most common cancer of the canine oral cavity resulting in significant morbidity and mortality. Recently a dog with multiple oral SCCs that contained a novel papillomavirus (PV) was reported. The aim of the present study was to determine the genome of this novel PV. To do this a short section of PV DNA was amplified from an oral SCC and ‘back-to-back’ primers were designed. Due to the circular nature of PV DNA, these primers were then used to amplify the remainder of the genome by inverse PCR. The PCR product was sequenced using next generation sequencing and the full genome of the PV, consisting of 8007 bp, was assembled and analysed. As this is the seventeenth PV identified from the domestic dog, the novel PV was designated Canis familiaris papillomavirus (CPV) type 17. Similar to other CPV types, the putative coding regions of CPV-17 were predicted to produce 5 early and 2 late proteins. Phylogenetic analysis of ORF L1 revealed greater than 70% similarity to CPV-2 and CPV-7 and we propose that CPV-17 also be classified as a Taupapillomavirus 1. While it appears CPV-17 is only rarely present in canine oral SCCs, evidence suggests that this PV could influence the development of oral SCCs in this species.

Spermatogenesis in animals is the process by which male spermatogonia develop into mature spermatozoa. In most taxa, the process involves changes in the basic proteins associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines producing compact packaging of the genome. Sperm chromatin in the Chinese mitten crab (Eriocheir sinensis) has a noncompacted loosely arranged organization. However, its formation during spermatogenesis is not clear. In this study, a cDNA sequence encoding histone H2B was cloned by polymerase chain reaction amplification, and its recombinant protein was expressed and purified. Protein alignment studies demonstrated that this histone H2B had 80.80%, 95.12%, 80.16%, 91.87%, 81.75%, 77.78% and 99.19% identity with its counterparts in zebrafish, fruit fly, human, prawn, mouse, African clawed frog, and crayfish, respectively. Western blotting indicated that the recombinant protein could be recognized by an anti-H2B antibody and confirmed that histone H2B exists in sperm nuclei. Immunofluorescence demonstrated that histone H2B was present in the nuclei of spermatogonia, spermatocytes, spermatids, and mature spermatozoa. This is the first report that the mature sperm nucleus of E. sinensis contains histone H2B. This work complements a previous study of sperm histones of this species and provides a basis for further study of the noncondensed sperm nuclei of decapod crustaceans.

Feline sarcoids are rare mesenchymal neoplasms of domestic and exotic cats. Previous studies have consistently detected short DNA sequences from a papillomavirus (PV), designated feline sarcoid-associated papillomavirus (FeSarPV), in these neoplasms. The FeSarPV sequence has never been detected in any non-sarcoid sample from cats but has been amplified from the skin of cattle suggesting that feline sarcoids are caused by cross-species infection by a bovine papillomavirus (BPV). The aim of the present study was to determine the genome of the PV that contains the FeSarPV sequence. Using the circular nature of PV DNA, four specifically designed ‘outward facing’ primers were used to amplify two approximately 4,000 bp DNA segments from a feline sarcoid. The two PCR products were sequenced using next generation sequencing and the full genome of the PV, consisting 7,966 bp, was assembled and analysed. Phylogenetic analysis revealed the PV was closely related to the species 4 delta BPVs-1, -2, and -13, but distantly related to any carnivoran PV genus. These results are consistent with feline sarcoids being caused by a BPV type and we propose a classification of BPV-14 for this novel PV. Initial analysis suggests that, like other delta BPVs, the BPV-14 E5 protein could cause mesenchymal proliferation by binding to the platelet derived growth factor beta receptor. Interestingly BPV-14 has not been detected in any equine sarcoid suggesting that BPV-14 has a host range that is limited to bovids and felids.