Identification of gonadal tissue in cryptorchid stallion can be improved by molecular biological analysis - a case report.

Surgically removed testicular tissue in cryptorchid stallions is sometimes difficult to identify because of morphological and histological malformation. Therefore, a sure method to characterise the removed tissue is required. A 2-year-old Haflinger stallion was castrated after diagnosis of cryptorchidism to remove the left intra-abdomnial testis. Intra-operative exploration of the abdominal cavity revealed a firm, dysmorphic structure, which could not be identified as testis based on macroscopic anatomy. The removed tissue was Bouin-fixed and paraffin-embedded for histological examination. We additionally applied immuno-histochemistry for smooth muscle actin to identify tubular structures as well as reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of steroidogenic acute regulatory protein (StAR), indicating the presence of Leydig cells. A hCG test was conducted after surgery to screen for remaining testicular tissue. Histological examination using haematoxylin and eosin staining revealed signs of tissue calcification, amorph matrix with scattered cells and round structures. The latter could not be definitely identified as tubules. Actin staining revealed a few tubular wall structures. StAR mRNA expression indicated the presence of Leydig cells in parts of the removed tissue. The hCG test after castration showed no increase in testosterone. Histological and molecular biological examination of extirpated tissue in cryptorchid stallions can play an important role in the identification of the malformed testes like structures. The use of molecular biological techniques provides the opportunity to characterise surgically removed abdominal tissue that cannot be clearly diagnosed by routine histological examination.

A case of equine granulocytic ehrlichiosis provides molecular evidence for the presence of pathogenic anaplasma phagocytophilum (HGE agent) in Germany.

Based on seroprevalence studies and tick infection rates, tick-borne human granulocytic ehrlichiosis (HGE) is thought to occur in Germany, but to date no clinical case has been detected. Reported here are the first ehrlichial sequences derived from a German horse that fell ill with granulocytic ehrlichiosis. The analysis of three different genes (16S rRNA gene, groESL, and ankA) revealed up to 100% identity with ehrlichial sequences derived from patients with HGE in other countries or from infected ticks in Germany. Thus, the current lack of clinical cases of HGE in Germany is unlikely to result from the absence of pathogenic granulocytic ehrlichiae strains in German ticks.

Dependence of yeast pre-mRNA 3'-end processing on CFT1: a sequence homolog of the mammalian AAUAAA binding factor.

3'-End formation of pre-mRNA in yeast and mammals follows a similar but distinct pathway. In Saccharomyces cerevisiae, the cleavage reaction can be reconstituted by two activities called cleavage factor I and II (CFI and CFII). A CFII component, designated CFT1 (cleavage factor two) was identified by its sequence similarity to the AAUAAA-binding subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF), even though the AAUAAA signal sequence appears to play no role in yeast pre-mRNA 3' processing. Depletion of a yeast whole-cell extract with antibodies to CFT1 protein abolished cleavage and polyadenylation of pre-mRNAs. Addition of CFII restored cleavage activity, but not polyadenylation. Polyadenylation required the further addition of poly(A) polymerase and polyadenylation factor I, suggesting a close but not necessarily direct association of these two factors with the CFT1 protein.

Pre-mRNA topology is important for 3'-end formation in Saccharomyces cerevisiae and mammals.

Various signal motifs that are required for efficient pre-mRNA 3'-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3'-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3'- and 5'-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3'-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3'-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3'-end formation is discussed.

PTF1 encodes an essential protein in Saccharomyces cerevisiae, which shows strong homology with a new putative family of PPIases.

Complementation of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae resulted in the isolation of PTF1 (processing/termination factor 1), an essential gene encoding a putative 3'-end processing or transcription termination factor of pre-mRNAs. Ptf1p shows significant homology to a newly discovered family of PPIases. This family is characterized by its insensitivity to immunosuppressive drugs and the lack of homology with cyclophilins and FK-506 binding proteins [Rahfeld et al. (1994) FEBS Lett. 352, 180-184]. Should Ptf1p display PPIase activity, it would be the first characterized, eukaryotic member of this putative family, which is essential for growth.

Flexibility and interchangeability of polyadenylation signals in Saccharomyces cerevisiae.

Various signal motifs have been reported to be essential for proper mRNA 3'-end formation in the yeast Saccharomyces cerevisiae. However, none of these motifs has been shown to be sufficient to direct 3'-end processing and/or transcription termination. Therefore, several structural motifs have to act in concert for efficient 3'-end formation. In the region upstream of the three polyadenylation sites of the yeast gene for alcohol dehydrogenase I (ADH1), we have identified a hitherto unknown signal sequence contained within the octamer AAAAAAAA. This motif, located 11 nucleotides upstream of the first ADH1 polyadenylation site, is responsible for the utilization of this site in vitro and in vivo, since mutational alteration drastically reduced 3'-end formation at this position. Insertion of 38 ADH1-derived nucleotides encompassing the (A)8 motif into the 3'-end formation-deficient cyc1-512 deletion mutant restored full processing capacity in vitro. Insertion of the octamer alone did not restore 3'-end formation, although mutation of the (A)8 motif in the functional construct had abolished 3'-end processing activity almost completely. This demonstrates that the sequence AAAAAAAA is a necessary, although not sufficient, signal for efficient mRNA 3'-end formation in S. cerevisiae.