The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

Meiosis in human spermatocytes has been analyzed by three dimensional reconstructions of 4 leptotene, 4 earlymid zygotene, 10 late zygotene, and 21 early pachytene nuclei. At leptotene, a lateral component is organized along each chromosome and the telomeres attach to the nuclear envelope. At early zygotene, the attachment sites aggregate and a chromosome bouquet is formed. Pairing and synaptonemal complex formation are initiated from the telomeres by binding of precursor material for the central region to the lateral components of the aligned homologues. In the 10 late zygotene nuclei, on the average 72% of the autosomal complement had been paired. Synaptonemal complex formation is in most cases initiated from both ends of the homologues and only in 5 cases was initiation of complex formation interstitial. Pairing of the short arms of the acrocentric bivalents and the X and Y chromosomes is delayed compared to the remainder of the genome. Irregularities such as interlockings and breaks of the lateral components of chromosomes or breaks of the synaptonemal complexes of bivalents are observed in 8 out of the 10 nuclei. Most of the breaks appeared to be the result of a resolution of interlockings. At early pachytene, all bivalents are fully paired the only exception being the secondary constrictions on bivalents 1 and 9 which frequently remain unpaired. In all nuclei, a short stretch of synaptonemal complex is present between the X and Y chromosomes. Only two interlockings and three breaks of lateral components of chromosomes were found at this stage. Recombination nodules are present in or at the central region of the synaptonemal complex from early zygotene and evidence has been obtained in favor of an attachment of nodules to precursor material for the central region at the pairing fork. Nodules can, however, also attach to a fully formed synaptonemal complex. At late zygotene, an average number of 101 nodules per nucleus is present. Assuming that all regions of the complex have equal probabilities of receiving a nodule about 144 nodules are expected to be present at the moment of complete pairing. At early pachytene, the mean number of nodules is 75. Generally, nodules appear to be distributed evenly along the bivalent arms. Nodules are present, however, in excess in the telomere regions and in the XY bivalent while the centromere regions, the secondary constrictions, and the short arms of the acroeentric bivalents are relatively depleted of nodules. Measurements of the distances between adjacent nodules and a comparison with a theoretical distribution of such distances assuming a random positioning of nodules have demonstrated that at late zygotene nodules are more frequently clustered than would be expected if they were distributed independently. At early pachytene, the reverse pattern is observed.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (⌬per9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced ⌬per9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.Peroxisomes are cell organelles that are present in virtually all eukaryotic cells. They perform specific metabolic functions that are often related to the developmental stage and/or the organism in which they occur (1). The metabolic importance of peroxisomes in humans is demonstrated by the fact that the absence of the organelles leads to severe abnormalities, followed by an early death (e.g. Zellweger syndrome (2)). Consequently, many studies are now devoted to unravel the molecular mechanisms of peroxisome biogenesis and function. Yeasts are excellent model systems for such studies having the advantages that (i) the induction and protein composition of peroxisomes can readily be manipulated by varying growth conditions and (ii) in the absence of peroxisomes, yeasts are viable (3, 4). Hence, peroxisome-deficient mutants have been isolated from different yeast species (4), and the corresponding genes are being cloned and characterized.In yeast, peroxisomes normally develop by growth and fission from pre-existing ones. Peroxisomal matrix proteins are nuclear-encoded, synthesized in the cytoplasm, and directed to the organelle by topogenic signals (PTSs).1 Two PTSs have been identified and are located either at the extreme C terminus (PTS1) or the N terminus of the protein (PTS2) (4). Our knowledge on the sorting of peroxisomal membrane proteins is still limited, and consensus topogenic sequences have yet to be identified (5).In our laboratory, we use the methylotrophic yeast Hansenula po...

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Serial sectioning followed by three dimensional reconstruction of lateral componenets of the synaptoemal complex have been used to follow chromosome pairing during theprophase of the achiasmatic meiotic division in the silkworm. Bombyx mori. During leptotene and early zygotene, the lateral components become attached to the nuclear envelope at a specific region, thus forming a chromosome bouquet. The attachment of lateral componenets to the nuclear envelope precedes the completion of the components between their attachment points. Synapsis and synaptonemal complex formation start during the period of lateral component organization in the individual nucleus. Telomeric movements on the nuclear envelope occur at two stages of the prophase: the chromosome pairing appears to be initiated by an association of unpaired ends of homologus chromosomes, the nature of this primary attraction and recognition being unknown. Secondly, the paired chromosomes become dispersed in the nucleus by shifting of attachment sites of completed synaptonemal complexes at the end of zygotene. This movement is possible related to a membranes flow occuring during this stage. Membrane material is synthesized at the region of synaptonemal complex attachment. Later, the excess membrane material is shifted to the opposite pole where it protudes into the lumen of the nuclei thus forming vacuoles. Two previously undescribed features of chromosomes paring were revealed. In late zygotene, chromosome pairing and synaptonmal complex formation were frequently observed to be delayed or even prevented over s short distance by interlocking two bivalents, both being attached to the nuclear envelope. Such interlocking of bivalents was not found in pachytene...

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14‐1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome‐inducing growth conditions. In cells of both pex14‐1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild‐type H.polymorpha cells resulted in a peroxisome‐deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.

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