Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs.

We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.     

Scanning and competition between AGs are involved in 3'' splice site selection in mammalian introns.

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.     

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.     

Computer prediction of the exon-intron structure of mammalian pre-mRNAs.

A novel approach to the problem of prediction of protein-coding regions is suggested. This approach combines the site prediction methods to predict splicing sites and the global coding region prediction methods to choose the best variant of spliced mRNA. One of the advantages of the suggested algorithm is that the resulting mRNA or protein sequence may then be immediately analyzed further. The true mRNA either coincides with the predicted one or ranks high in the list of variants. In the latter situation the predicted mRNA usually differs from the true one in only one or two of several exons. The combined approach allows the use of a priori information (e.g. the putative protein length or the number of exons). It is possible to use additional parameters not considered here, such as the preferred lengths of exons and introns, and particularly the preferred position of introns in the reading frame and the preferred codon position of exon termini.     

Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes.

In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA. One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC. Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro. Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint. These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs. Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.     

Interactions between introns via exon definition in plant pre-mRNA splicing

The barley gene Mlo encodes a prototype of a novel class of plant proteins. In mlo mutants, absence of the 60 kDa wild-type Mlo protein results in broad-spectrum resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei . To directly assess its function, Mlo was transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells of mlo resistant barley lines. Fungal inoculation of epidermal cells transfected with wild-type Mlo led to haustorium formation and abundant sporulation. Therefore, expression of the wild-type Mlo gene, in mlo resistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation of mlo resistance alleles was restricted to single host cells, indicating a cell-autonomous function for the wild-type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide-ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack.     

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs

We used nuclear extracts from Drosophila Kc cells to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila SLBP plays a critical role in recruiting the U 7 snRNP to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the HDE does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U 7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U 7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U 7 snRNA was not able to deplete the U 7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U 7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates.     

Interactions between pairs of DNA-specific fluorescent stains bound to mammalian cells.

The interactions between DNA-specific fluorescence stains complexed with mitotic Chinese hamster cells were studied by spectrofluorometric and flow fluorometric techniques. The degree of binding interactions and of energy transfer between stains was determined from the intensities and shapes of fluorescence emission spectra of cells complexed with pairs of stains. The stain pairs Hoechst 33258-chromomycin A3, Hoechst 33258-ethidium bromide, and chromomycin A3-ethidium bromide exhibited efficient energy transfer from the short wavelength absorber (donor) to the long wavelength absorber (acceptor), and little competitive or cooperative binding of stains. The stain pair quinacrine-ethidium bromide exhibited both energy transfer and competitive binding. None of the stain pairs showed evidence of strong electronic interactions between stains. The magnitude of energy transfer interactions was used to estimate the quantity and distribution of the stains molecules complexed to mitotic cells. The results indicate a fairly even distribution of each of these stains along the DNA of intracellular mitotic chromosomes.     

Interactions between nucleic acid bases. New parameters of potential functions and energy minima

The parameters of atom-atom potential functions suggested by one of the authors in 1979-1986 were slightly changed. The changes were performed to achieve a better agreement with experimental data of interaction energy values in global minima and hydrogen bond lengths. These changes resulted in better accord with experimental values of distances between the layers in DNA monomer crystals and between the base pairs in oligonucleotide duplexes. The refined potential functions were used to calculate the energy of interactions between nucleic acid bases in various mutual positions. The calculations revealed a few types of mutual base arrangements in minima of interaction energy for each pairwise base combination. A new type of minima was found, which correspond to a nearly perpendicular arrangement of base rings and the formation of the intermolecular hydrogen bond.     

Syntaxin 2 and endobrevin are required for the terminal step of cytokinesis in mammalian cells

The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.     

Interactions between nucleotide bases in coplanar and stacking dimers under vacuum. Mass spectrometric study

New temperature-dependent field--ionization mass-spectrometric method is described. It allows to measure enthalpies of associates in vacuum. An analysis of all the experimental results led to a conclusion that interactions in coplanar dimers of nucleic acid bases are stronger than in the stacking ones. A comparison between the vacuum measurements and theoretical data is made.     

Interactions between SRY and SOX genes in mammalian sex determination

The SRY gene on the mammalian Y chromosome undoubtedly acts to determine testis, but it is still quite unclear how. It was originally supposed that SRY acts directly to activate other genes in the testis-determining pathway. This paper presents an alternative hypothesis that SRY functions indirectly, by interacting with related genes SOX3 (from which SRY evolved) and SOX9 (which appears to be intimately involved in vertebrate gonad differentiation). Specifically, I propose that in females SOX3 inhibits SOX9 function, but in males, SRY inhibits SOX3 and permits SOX9 to enact its testis-determining role. This hypothesis makes testable predictions of the phenotypes of XX and XY individuals with deficiencies or overproduction of any of the three genes, and is able to account for the difficult cases of XX(SRY−) males and transdifferentiation in the absence of SRY. The hypothesis also suggests a way that the dominant SRY sex-determining system of present-day mammals may have evolved from an ancient system relying on SOX3 dosage. BioEssays 20 :264–269, 1998. © 1998 John Wiley & Sons, Inc.     

The terminal DNA structure of mammalian chromosomes.

In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.     

Reading two bases twice: mammalian antizyme frameshifting in yeast.

Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30-fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal.