Tsix, a gene antisense to Xist at the X-inactivation centre.

In mammals, dosage compensation is achieved by X inactivation and is regulated in cis by the X-inactivation centre (Xic) and Xist. The Xic controls X-chromosome counting, choice of X to inactivate and initiation of silencing. Xic action culminates in a change in Xist RNA property from a scarce, unstable RNA to highly expressed Xist RNA that coats the future inactive X. Deleting a 65-kb region downstream of Xist results in constitutive Xist expression and X inactivation, implying the presence of a cis-regulatory element. In this region, we now report the discovery of a gene antisense to Xist. Tsix is a 40-kb RNA originating 15 kb downstream of Xist and transcribed across the Xist locus. Tsix sequence is conserved at the human XIC. Tsix RNA has no conserved ORFs, is seen exclusively in the nucleus and is localized at Xic. Before the onset of X inactivation, Tsix is expressed from both X chromosomes. At the onset of X inactivation, Tsix expression becomes monoallelic, is associated with the future active X and persists until Xist is turned off. Tsix is not found on the inactive X once cells enter the X-inactivation pathway. Tsix has features suggesting a role in regulating the early steps of X inactivation, but not the silencing step.

An improved fragile X Southern blot probe without the CGGs eliminates background bands.

The largest of the commonly used probes for Southern blot diagnosis of fragile X mental retardation syndrome spans the CGG repeat cluster in the FMR-1 gene. This probe causes the appearance of 'common' or 'constant' background bands which occasionally complicate the interpretation of autoradiographic results. By removing a 357 bp Sphl to Nhel fragment containing the CGGs from the probe pE5.1, we constructed a probe which eliminates the background bands yet allows the use of a large probe (4.8 kb) to detect changes in the diagnostic 5.2 kb genomic EcoRl band. This CGG-deficient probe has been used in routine diagnostic cases as well as in second round testing of pE5.1-probed cases where enlarged mutant bands are suspected to comigrate with the background bands.

A simple fragile X PCR assay with 7-deazaguanine-substituted DNA visualized by ethidium bromide.

A deazaguanine-substituted DNA PCR product from FMR-1 (the fragile X mental retardation syndrome gene) can be efficiently visualized with ethidium bromide on standard agarose gels. Normal-sized alleles (less than 54 CGG repeats) generated strong, easily visible bands in the expected size range of 491-635 bp. Southern blot analysis and radioactive PCR on sequencing gels were used to verify that the 74 males (out of 245 total tested) whose DNA failed to generate a visible band contained premutations or full mutations. This technique can be used as an inexpensive screen for fragile X syndrome among developmentally delayed males.

Mutations affecting the activity of urokinase-type plasminogen activator.

Mutagenesis throughout the single-chain urokinase-type plasminogen activator (scu-PA) cDNA molecule, followed by expression of the mutant genes and secretion of the resulting mutant proteins from yeast, has been used to determine the amino acid residues important for activity of scu-PA molecules. Twelve out of 13 colonies secreting variant scu-PA molecules with decreased ability to form a zone of fibrinolysis had mutant genes with a single codon alteration in the serine protease encoding domain (B-chain). Many of these changes are of highly conserved residues in the serine proteases and are consequently of considerable interest. A model three-dimensional structure of the protease domain of urokinase was used to explain the basis for the effects of these down mutations. The model showed that the strongest down mutations result from either interference of the mutated side chain with substrate binding at the active site or the introduction of bulky or charged groups at structurally sensitive internal positions in the molecule. Attempts to find second site revertants of five down mutants, altered either at the plasmin activation site or near the serine at the active site, only resulted in same-site revertants, with the original or closely related amino acids restored.

Production of proteins by secretion from yeast.

To summarize, a variety of stable vectors and efficient promoters and secretion signals are available in yeast for engineering the secretion of any protein of interest. Since secretion is growth-associated, we have favored the use of constitutive promoters and moderate copy number integrated vectors. This is because (1) heterologous gene expression from very high copy number vectors is frequently deleterious to growth and (2) delaying gene expression until after the most rapid cell growth phase is cumbersome on a large scale. Methods are available for dividing the total process into growth and production/secretion phases, but they appear worthwhile only when expression of the engineered protein compromises growth significantly. Even with these useful tools, it is frequently helpful to enlist the aid of mutant host strains in order to maximize secretion of a desired protein. Mutations in the PMR1 gene have proved effective in a number of different cases. Moreover, it is possible to identify new host strains tailored to specific needs by applying activity screens to mutagenized colonies growing on petri plates. Finally, colony screens such as the ones described here for active secreted enzymes are useful for routine strain construction. For example, they may be applied to identify the most productive strain from a large number of clones following a transformation or genetic cross. In addition, these screens may be used for characterizing the products of random mutagenesis of the gene encoding the secreted enzyme. The resulting structure-function information can be used to identify regions of the enzyme involved in different activities and to build new enzymes with different characteristics.

The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family.

The genes for two new P-type ATPases, PMR1 and PMR2, have been identified in yeast. A comparison of the deduced sequences of the PMR proteins with other known ion pumps showed that both proteins are very similar to Ca2+ ATPases. PMR1 is identical to SSC1, a gene previously identified by its effect on secretion of some foreign proteins from yeast. Proteins secreted from pmr1 mutants lack the outer chain glycosylation that normally results from passage through the Golgi. Loss of PMR1 function suppresses the lethality of ypt1-1, a mutation that blocks the secretion pathway. These data suggest that PMR1 functions as a Ca2+ pump affecting transit through the secretory pathway.

Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.

The Yarrowia lipolytica LEU2 gene.

A 2810 bp DNA fragment containing the beta-isopropylmalate dehydrogenase gene of the dimorphic yeast Yarrowia lipolytica has been sequenced. The sequence contains an open reading frame of 405 codons, predicting a protein of 43,366 molecular weight. Protein sequence homology with the polypeptide encoded by the LEU2 gene of Saccharomyces cerevisiae is 64%, whereas DNA sequence homology is 61%. The 5'- and 3'-flanking regions of the Y. lipolytica LEU2 gene share only some general structural features common to genes of S. cerevisiae such as the presence and location of TATA boxes, CAAT boxes, CACACA repeats, the lack of G residues in the 5'-untranslated region and 3'-transcription terminators. Transcription of a 1.4 kb mRNA begins at a small cluster of sites approximately 40 base pairs before the initial ATG.

Cloning and characterisation of the ribosomal RNA genes of the dimorphic yeast, Yarrowia lipolytica.

The ribosomal RNA genes of Yarrowia lipolytica have been identified, both in restriction digests of total genomic DNA and in a pBR322 gene bank, by hybridisation with cloned Saccharomyces cerevisiae rDNA. The Y. lipolytica rDNA repeat unit is 8.9 kb in size and contains the genes for the 25S and 18S, but not the 5S, rRNA species. The number of copies of these repeat units is approx. 50 per haploid genome. Several clones were found which did not conform to the standard restriction map due to differences outside the coding region. It appears that there is either heterogeneity of the spacer sequence within a strain or that the Y. lipolytica rDNA genes may be present as a number of separate clusters within this yeast's genome.

Enhanced gene conversion and postmeiotic segregation in pachytene-arrested Saccharomyces cerevisiae.

Previous study has demonstrated that incubation of yeast cells of strain AP-1 in sporulation medium at 36 degrees permits them to begin meiosis but that they become arrested at pachytene and undergo enhanced intragenic recombination between ade2 heteroalleles. Tetrad analysis was undertaken to characterize the altered program of meiotic recombination more widely. In one set of experiments, pachytene-arrested cells were permitted to resume sporulation upon transfer to the permissive temperature. In the resulting asci, both postmeiotic segregation and gene conversion were increased several-fold at a number of loci relative to unarrested controls, whereas reciprocal recombination increased two- to threefold. Another set of experiments analyzed the genetic consequences of inducing the pachytene-arrested cells to revert directly to mitotic growth without completion of meiosis. The appearance of homozygous sectors from heterozygous markers revealed that these cells had become committed to appreciable recombination but that reciprocal exchange was less frequent than in normal asci. Taken together, the data indicate that pachytene arrest rendered the cells committed to enhanced recombination upon resumption of sporulation but that most of the crossing over did not occur until release from the arrest.--The genetic basis of pachytene arrest by AP-1 was investigated by mating each of its parents with progeny of strain Y55, which is able to sporulate at 36 degrees. Both of these diploids sporulated at 36 degrees, and asci from the one studied further exhibited 2:2 segregation of the sporulation defect, indicating that pachytene arrest is dependent on a recessive, temperature-sensitive allele at a chromosomal locus.

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.

Preferential Occurrence of Nonsister Spores in Two-Spored Asci of SACCHAROMYCES CEREVISIAE: Evidence for Regulation of Spore-Wall Formation by the Spindle Pole Body.

Yeast cells subjected to a reversible thermal arrest of meiosis yielded progressively fewer spores per ascus as the arrest was extended. Dissection of two-spored asci by a newly developed method that prevents selection of false asci revealed that the spores were not a random sample of the haploid meiotic products. Most, if not all, pairs of spores contain nonsister products of the reductional division. Electron microscopic examination of the meiotic cells revealed the cytological basis for this bias. All four spindle pole bodies (SPBs) present at the second meiotic division normally gain a structural modification (the outer plaque) upon which the initiation of the prospore wall occurs. In the formation of a two-spored ascus, only one spindle pole body on each meiosis II spindle was so modified. These observations suggest that the morphogenesis of spores is regulated at meiosis II by limiting the number of SPBs gaining the outer plaque. The enhancement of spore yield upon addition of fresh medium suggests that this morphogenetic regulation responds more directly to nutrient deprivation arising during the thermal arrest, rather than to elevated temperature per se.