Right End Of Chromosome Research Articles

Targeted mutagenesis of the ring‐exported protein‐1 of Plasmodium falciparum disrupts the architecture of Maurer's cleft organelles

Mature red blood cells have no internal trafficking machinery, so the intraerythrocytic malaria parasite, Plasmodium falciparum, establishes its own transport system to export virulence factors to the red blood cell surface. Maurer's clefts are parasite-derived membranous structures that form an important component of this exported secretory system. A protein with sequence similarity to a Golgi tethering protein, referred to as ring-exported protein-1 (REX1), is associated with Maurer's clefts. A REX1-GFP chimera is trafficked to the Maurer's clefts and preferentially associates with the edges of these structures, as well as with vesicle-like structures and with stalk-like extensions that are involved in tethering the Maurer's clefts to other membranes. We have generated transfected P. falciparum expressing REX1 truncations or deletion. Electron microscopy reveals that the Maurer's clefts of REX1 truncation mutants have stacked cisternae, while the 3D7 parent line has unstacked Maurer's clefts. D10 parasites, which have lost the right end of chromosome 9, including the rex1 gene, also display Maurer's clefts with stacked cisternae. Expression of full-length REX1-GFP in D10 parasites restores the 3D7-type unstacked Maurer's cleft phenotype. These studies reveal the importance of the REX1 protein in determining the ultrastructure of the Maurer's cleft system.

Chromosome XII context is important for rDNA function in yeast.

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of ∼150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6–12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae.

Minichromosome formation in Chlorella cells irradiated with electron beams

Three minichromosomes, miniP7, miniB7, and miniK4 of 800 kbp, 450 kbp, and 550 kbp, respectively, were obtained from Chlorella vulgaris chromosome I by electron-beam irradiation. Two of them were structurally characterized: MiniP7 was formed by the deletion of an internal 180 kbp close to the right end of chromosome I. The 180-kbp region with a small interspersed nuclear element (SINE)-like element on its left terminus was translocated to another chromosome, leaving a footprint-like structure on miniP7. MiniB7 was a hybrid of chromosome I and another chromosome, retaining the left telomere and the centromere of chromosome I. The centromeric repetitive elements served as a rearrangement point in the miniB7 formation. These examples showed the complicated mechanisms involved in the minichromosome formation. The minichromosomes thus obtained can be useful for isolating the fundamental structural elements of a chromosome. Moreover, they may serve as starting materials or a vector to generate artificial chromosomes carrying useful genes.

Minichromosome Formation in Chlorella Cells Irradiated with Electron Beams

Three minichromosomes, miniP7, miniB7, and miniK4 of 800 kbp, 450 kbp, and 550 kbp, respectively, were obtained from Chlorella vulgaris chromosome I by electron-beam irradiation. Two of them were structurally characterized: MiniP7 was formed by the deletion of an internal 180 kbp close to the right end of chromosome I. The 180-kbp region with a small interspersed nuclear element (SINE)-like element on its left terminus was translocated to another chromosome, leaving a footprint-like structure on miniP7. MiniB7 was a hybrid of chromosome I and another chromosome, retaining the left telomere and the centromere of chromosome I. The centromeric repetitive elements served as a rearrangement point in the miniB7 formation. These examples showed the complicated mechanisms involved in the minichromosome formation. The minichromosomes thus obtained can be useful for isolating the fundamental structural elements of a chromosome. Moreover, they may serve as starting materials or a vector to generate artificial chromosomes carrying useful genes.

The cytoadherence linked asexual gene family of Plasmodium falciparum: are there roles other than cytoadherence?

The binding of erythrocytes infected with Plasmodium falciparum to the endothelium lining the small blood vessels of the brain and other organs can mediate severe pathology. A region at the right end of chromosome 9 has been implicated in the binding of parasitised erythrocytes to the endothelial receptor CD36. A gene expressed in asexual erythrocytic stage parasites has been identified in this region and termed the cytoadherence linked asexual gene (clag). Antisense RNA production and targeted gene disruption of clag resulted in greatly reduced binding to CD36. Hybridisation to 3D7 chromosomes showed clag to be a part of a gene family of at least nine members. All members analysed so far have a conserved gene structure of at least nine exons, as well as putative transmembrane domains. The possible functions of the gene family are discussed.

Disruption of a novel open reading frame of Plasmodium falciparum chromosome 9 by subtelomeric and internal deletions can lead to loss or maintenance of cytoadherence

Isolates of Plasmodium falciparum commonly undergo a large subtelomeric deletion of the right end of chromosome 9 during in vitro cultivation, accompanied by loss of ability to cytoadhere to melanoma cells and greatly lowered gametocyte production. ItG2, an isolate in which cytoadherence is stable, has undergone a subtelomeric deletion of intermediate length on chromosome 9. We show here that the deletions in all non-cytoadherent clones examined have breakpoints within or delete a novel open reading frame (the breakpoint open reading frame, BPORF) that is a unique sequence in the genome. Surprisingly, in ItG2 BPORF has been removed by an additional 15 kb internal deletion in chromosome 9. These results suggest mechanisms to account for the observed frequent deletion of the right arm of chromosome 9 and for the known stability of cytoadherence in ItG2. However, we were unable to detect var genes in this region of the ItG2 genome. We conclude that the product of a novel gene distinct from the var family is implicated in cytoadherence.

Positional cloning of a sequence from the breakpoint of chromosome 9 commonly associated with the loss of cytoadherence.

Isolates of Plasmodium falciparum commonly undergo a large, subtelomeric deletion of the right end of chromosome 9 during in-vitro cultivation. This deletion is usually accompanied by loss of ability to cytoadhere to melanoma cells, loss of a var-gene product from the red-cell surface and a reduction in gametocyte production. However, cytoadherence is stable in the isolate ItG2, remaining after many generations in culture. Deletions in all the non-cytoadherent clones examined have breakpoints within or delete a novel open-reading frame, called the breakpoint open-reading frame (BPORF), that is a unique sequence in the genome. In ItG2, surprisingly, BPORF has been removed by a 15-kb deletion, internal in chromosome 9. These results indicate mechanisms to explain why the deletion of chromosome 9 occurs so frequently and why cytoadherence is stable in ItG2.

Genome Structure, Mapping and Expression of the Tropomyosin Gene tmy-1of Caenorhabditis elegans

The complete tropomyosin gene, designated tmy-1, of Caenorhabditis eleganswas recovered by genome walking from a fragment that was obtained by exon-expression cloning using specific cloning using specific anti-tropomyosin antiserum as a probe. The genome structure of the tmy-1gene has been determined by combining the DNA sequences of cDNA clones with those of the genomic fragments. The single-copy gene spans approximately 13 kb and include 14 exons. Comparison of cDNA and genomic sequences demonstrates that three isoforms are encoded by the gene tmy-1. Homology of the 27 C-terminal amino acid residues to those of Drosophilaand vertebrates suggest that these may be the body wall, pharyngeal and non-muscle types. Tissue-specific expression of the tmy-1gene was determined by microinjection of a promoter/ lacZfusion gene and with immunohistochemistry by using affinity-purified tissue-specific anti-tropomyosins. The 5′ end promoter common to CeTMI and CeTMII is expressed in the body wall muscles, vulva, anus muscles and male tail muscles. Control sequences of the 5′ end promoter are located 660 to 800 bp upstream of the initial methionine codon. The third isoform, CeTMIII, encoding 256 amino acids residues was expressed in the pharyngeal muscles by the promoter in the third intron. The mRNA of CeTMIII was trans-spliced with SL1 and SL2. These results allow us to solve the question of what is common from this worm to vertebrates, and also what are the cross-species complexities and the tissue-specific differences of tropomyosins. The tmy-1gene is located on the C. elegansgenomic YAC grid near the right end of chromosome I, in the region on the lev-11gene.

Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13, 47-52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47, 2889-2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.

Transcriptional regulation of flocculation genes inSaccharomyces cerevisiae

Northern analysis showed that DNA from the flocculation gene FLO1 hybridized to mRNA molecules of 4.8 kb. This transcript was specific for the FLO1 gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing to FLO1 DNA, both in various flocculent and non-flocculent strains and in cells from the non-flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non-flocculating cultures. From these results we conclude that the FLO1 gene is transcriptionally regulated. Mutations in TUP1 or SSN6 cause flocculation. Several transcripts hybridizing to FLO1 DNA were present in the mutants but not in the corresponding wild-type strains. Disruption of the FLO1 gene in the tup1 and ssn6 strains showed that one of the transcripts corresponded to the FLO1 gene. Disruption of FLO1 did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, including FLO1, are activated or derepressed by mutations in the TUP1/SSN6 regulatory cascade.

ATP1 and ATP2, the F1F0-ATPase alpha and beta subunit genes of Saccharomyces cerevisiae, are respectively located on chromosomes II and X.

Southern blot analysis showed that ATP1 and ATP2 map on chromosomes II and X, respectively. Physical mapping of ATP1 and ATP2 by chromosome fragmentation showed that ATP1 is at the left end of chromosome II and ATP2 is at the right end of chromosome X. Both are located close to telomere sequences of each chromosome; ATP1 and ATP2 being approximately 30 kb and 85 kb from the respective telomeres.

Mapping the genetic locus implicated in cytoadherence of Plasmodium falciparum to melanoma cells

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro cultivation accompanied by loss of cytoadherence to melanoma cells. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling pfEMP1 expression is encoded in this region. Initially a library of short fragments highly enriched for the right arm of chromosome 9 was constructed in bacteriophage λ. Clones from this library were obtained randomly by the polymerase chain reaction (PCR) technique, sequenced and used to screen a yeast artificial chromosome (YAC)- P. falciparum library by PCR so that the region could be cloned and physically mapped in detail. We have used probes from this region to demonstrate that clones derived from ITG2 have undergone a deletion of intermediate length on chromosome 9. This could explain the unusual stability of cytoadherence in these clones

Isolation and characterization of SGE1: a yeast gene that partially suppresses the gal11 mutation in multiple copies.

Recessive mutations of GAL11 in Saccharomyces cerevisiae cause pleiotropic defects that include weak fermentation of galactose, alpha-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gal11P functions as a "co-activator" that links transcriptional activators, such as Gal4p and Grf1p/Rap1p/Tuf1p, with the basic transcription machinery. In the present experiments we isolated a gene, SGE1, that suppresses gal11 for growth on ethidium bromide/galactose agar when the gene was present in two or more copies. The other gal11 phenotypes were not suppressed by SGE1 in the multiple-copy state. Multiple copies of SGE1 increased expression of galactose-inducible genes in gal11 yeast, presumably at the level of transcription. When SGE1 was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGE1 encodes a predicted protein of 543 amino acids. SGE1-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame. The gene mapped near RAD56, at the right end of chromosome 16.

Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae.

The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry. To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned. The identity of the gene was confirmed by the non-flocculent phenotype of cells in which the C-terminal part of the gene had been replaced by the URA3 gene. Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I. A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated. Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a non-flocculent strain. The 3' end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere-proximal to PHO11. Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

A yeast origin of replication is activated late in S phase

The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: localization of a repeated sequence containing an acid phosphatase gene near a telomere of chromosome I and chromosome VIII.

A 17 kb region from near the right end of chromosome I of Saccharomyces cerevisiae was isolated on recombinant lambda bacteriophages. This region contained the PHO11 gene which was located only 3.4 kb from the right end of the chromosome. We found that this region also was repeated approximately 13 kb from the end of the chromosome VIII DNA molecule. The chromosome VIII sequence appears to be a previously unnamed acid phosphatase gene that we propose to call PHO12. Thus, similar to the repeated SUC, MAL, X and Y' sequences, some members of the repeated acid phosphatase gene family also appear near the termini of yeast chromosomes.

Cloning and molecular characterization of a telomeric sequence from a temperature-induced Balbiani ring

In Chironomus thummi thummi a Balbiani ring-like structure is induced at the right end of chromosome III by heat shock. Telomere-specific clones were obtained by microcloning this chromosome region. One of the recombinant clones, λCthS, which had an insert of 176 base pairs (bp), hybridized in situ to all the chromosome ends of C. th. thummi except one on the acrocentric chromosome IV. Homologous sequences were also present at two interstitial sites on chromosome II. Southern analysis of genomic DNA indicated that the 176-bp insert represents a basic repeating unit clustered in tandem arrays. Around 1000 copies of this sequence were located in each telomere. The repeats were homogeneous in size but showed some sequence divergence, which seemed to increase when different telomeres were compared. At the nucleotide sequence level the most noticeable feature was the presence of tracts of adenine alternating with tracts of thymine. In situ hybridzation to chromosomal RNA showed that the 176-bp repeat was complementary to the RNA transcribed at the T-BRIII region, and occasionally, to the RNA transcribed at some of the other telomeres during heat shock. Northern blot analysis of total RNA showed that a transcript larger than 10 kilobase pairs (kb) and complementary to the teloeric insert was present in heat-shocked as well as in untreated cells. However, at most only a very weak RNA hybridization was seen at the telomeres of untreated animals.

Sub-units of acid phosphatase-1 in Drosophila melanogaster: reversible dissociation in vitro.

THE genetic control of an acid phosphatase in the sibling species Drosophila melanogaster and D. simulans has been reported by one of us1. “Slow” and “fast” electrophoretic variants of this enzyme, called acid phosphatase-1 or Acph-1, are specified by codominant alleles, Acph-1A and Acph-1B, of a gene located very near the right end of chromosome 3 in each species. Zymograms of the heterozygotes (and interspecific hybrids) show three bands or zones of acid phosphataso activity: the “slow” and “fast” or Acph-1AA and Acph-1BB forms of the enzyme which are characteristic of the homozygous parents, and a third, more heavily staining zone which occupies a position between the parental enzymes.