Rad6-dependent ubiquitination of histone H2B in yeast.

Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae, and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.

Multiple and distinct activation and repression sequences mediate the regulated transcription of IME1, a transcriptional activator of meiosis-specific genes in Saccharomyces cerevisiae.

IME1 encodes a transcriptional activator required for the transcription of meiosis-specific genes and initiation of meiosis in Saccharomyces cerevisiae. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. Upon nitrogen depletion a transient induction in the transcription of IME1 is observed in MATa/MATalpha diploids but not in MAT-insufficient strains. In this study we demonstrate that the transcription of IME1 is controlled by an extremely unusual large 5' region, over 2,100 bp long. This area is divided into four different upstream controlling sequences (UCS). UCS2 promotes the transcription of IME1 in the presence of a nonfermentable carbon source. UCS2 is flanked by three negative regions: UCS1, which exhibits URS activity in the presence of nitrogen, and UCS3 and UCS4, which repress the activity of UCS2 in MAT-insufficient cells. UCS2 consists of alternate positive and negative elements: three distinct constitutive URS elements that prevent the function of any upstream activating sequence (UAS) under all growth conditions, a constitutive UAS element that promotes expression under all growth conditions, a UAS element that is active only in vegetative media, and two discrete elements that function as UASs in the presence of acetate. Sequence analysis of IME1 revealed the presence of two almost identical 30- to 32-bp repeats. Surprisingly, one repeat, IREd, exhibits constitutive URS activity, whereas the other repeat, IREu, serves as a carbon-source-regulated UAS element. The RAS-cyclic AMP-dependent protein kinase cAPK pathway prevents the UAS activity of IREu in the presence of glucose as the sole carbon source, while the transcriptional activators Msn2p and Msn4p promote the UAS activity of this repeat in the presence of acetate. We suggest that the use of multiple negative and positive elements is essential to restrict transcription to the appropriate conditions and that the combinatorial effect of the entire region leads to the regulated transcription of IME1.

Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1.

The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11 In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci.

The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance.

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.

Positive and negative feedback loops affect the transcription of IME1, a positive regulator of meiosis in Saccharomyces cerevisiae.

The IME1 gene of Saccharomyces cerevisiae encodes a transcription factor that is required for the expression of meiosis-specific genes. Like many of the genes it regulates, IME1 itself is expressed according to the following complex pattern: barely detectable levels during vegetative growth, and high induced levels under starvation conditions, followed by a subsequent decline in the course of meiosis. This report examines the influence of Ime1 protein on its own expression, demonstrating feedback regulation. Disruption of either IME1 or IME2 leads to constantly increasing levels of Ime1-lacZ expression, under meiotic conditions. This apparent negative regulation is due to cis elements in the IME1 upstream region, which confer transient meiotic expression to heterologous promoter-less genes. A specific DNA/protein complex, whose level is transiently increased under meiotic conditions, is detected on this element. In ime1- diploids, the level of this DNA/protein complex increases, without any decline. These results indicate that the transient expression of IME1 is apparently due to transcriptional regulation. This report also presents evidence suggesting that Ime1p is directly responsible for regulating its own transcription. Positive feedback regulation in mitotic conditions is suggested by the observation that overexpression of Ime1p leads to increased levels of IME1-lacZ. Negative autoregulation in meiotic cultures is demonstrated by the observation that a specific point mutation in IME1, ime1-3, permits expression of meiosis-specific genes, as well as induction of meiosis, but is defective in negative-feedback regulation of IME1.

IME1 gene encodes a transcription factor which is required to induce meiosis in Saccharomyces cerevisiae.

Previous studies have shown that the IME1 gene is required for sporulation and the expression of meiosis specific genes in Saccharomyces cerevisiae. However, sequence analysis has not revealed the precise functional role of the Ime1 protein. By engineering constructs which express various portions of the Ime1p fused to either the DNA binding or transcriptional activation domains of GAL4, we have conclusively demonstrated that IME1 is a transcription factor, apparently required for sporulation to activate the transcription of meiosis specific genes. The full Ime1p, when fused to the GAL4 DNA binding domain, can both activate GAL1-lacZ expression, and complement ime1-0 (a null allele) for the ability to sporulate, and transcriptionally activate IME2, a meiosis specific gene. As successively larger portions of the encoded Ime1p N-terminus are deleted from the GAL4(bd)-IME1 construct, the encoded fusion proteins retain the ability to complement an ime1 null allele, despite a decreasing ability to activate GAL1-lacZ transcription. However, a fusion construct which retains only the last 45 C-terminal amino acids of IME1 provides neither transcriptional activation of GAL1-lacZ nor complementation of ime1-0. Fusion of a GAL4 activation domain to this portion of IME1, results in a construct with a restored ability to complement an ime1-0 allele. This restored ability is dependent upon galactose induction. We conclude, therefore, that IME1 functions in meiosis as a transcriptional activator.

Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain.

Duchenne muscular dystrophy (DMD), a sex-linked degenerative disorder of the muscle, is one of the most common lethal genetic diseases in man. It affects about one male in 3,500, with an estimated one-third of cases being caused by new mutations. A less severe disease, Becker's muscular dystrophy (BMD), maps to the same chromosomal locus and is most probably an allelic form of DMD. Both diseases are sometimes associated with various degrees of mental retardation; the molecular basis of these phenotypes is unknown (for review, see ref. 1). The giant DMD gene spans approximately 2,000 kilobases (kb) (0.05% of the human genome) and encodes a 14-kb mRNA. The tissue-specificity of its expression has not been precisely determined. Monaco et al., using Northern blots, reported expression of the gene in human fetal skeletal muscle and small intestine but not in human fetal brain, or in human cultured myoblasts and transformed B and T cells. More recently, expression was detected in mouse skeletal and cardiac muscle, but not in mouse brain. Here we show, using a ribonuclease protection assay, that the DMD gene is developmentally regulated in rat and mouse myogenic cell cultures, and that it is expressed in rat and mouse striated muscle, in mouse smooth muscle and in rat, mouse and rabbit brain. We could not detect transcripts in other non-muscle tissues.