Jose M. Ortiz

Schizosaccharomyces pombe protein phosphatase 1 in mitosis, endocytosis and a partnership with Wsh3/Tea4 to control polarised growth.

PP1 holoenzymes are composed of a small number of catalytic subunits and an array of regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related catalytic subunits, Dis2 and Sds21. The gene for either protein can be individually deleted, however, simultaneous deletion of both is lethal. We fused enhanced green fluorescent protein (EGFP) coding sequences to the 5′ end of the endogenous sds21+ and dis2+ genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips and endocytic vesicles. This actin-dependent endocytosis occurred between nuclei and growing tips and was polarised towards growing tips. When dis2+ was present, Sds21.NEGFP was predominantly a nuclear protein, greatly enriched in the nucleolus. When dis2+ was deleted, Sds21.NEGFP levels increased and Sds21.NEGFP was then clearly detected at centromeres, endocytic vesicles and cell tips. Dis2.NEGFP was recruited to cell tips by the formin binding, stress pathway scaffold Wsh3 (also known as Tea4). Wsh3/Tea4 modulates polarised tip growth in unperturbed cell cycles and governs polarised growth following osmotic stress. Mutating the PP1 recruiting RVXF motif in Wsh3/Tea4 blocked PP1 binding, altered cell cycle regulated growth to induce branching, induced branching from existing tips in response to stress, and blocked the induction of actin filaments that would otherwise arise from Wsh3/Tea4 overproduction.

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements

SummaryA revised physical map of the α-haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.

Induction of apolipoprotein E gene expression in human and experimental atherosclerotic lesions

Hypercholesterolemia and atherosclerosis were induced in New Zealand White rabbits by cholesterol feeding. Apolipoprotein E mRNA levels in livers were found to be slightly increased, as determined by Northern blots. Apolipoprotein E gene expression was dramatically induced in rabbit atherosclerotic aortas with respect to healthy aortas. However, apolipoprotein E mRNA levels in atherosclerotic aortas were low as compared with the hepatic mRNA levels of the same animals. Interestingly, we also found a significant increase in apolipoprotein E expression in human atheromata with respect to healthy aorta from the same individual. This is the first report on apo E gene induction in human atherosclerotic lesions.

Analysis of IncF plasmids evolution: nucleotide sequence of an IncFIII replication region.

We determined the nucleotide sequence of RepFIII, the IncFIII replication region of the plasmid pSU316. Our data confirmed that RepFIII belongs to the RepFIIA family. The comparison of the nucleotide sequences from several RepFIIA-family plasmids revealed that pSU316 and R1 replicons are almost identical (96% similarity). Most of the differences between them are clustered in the incompatibility determinant. Analysis of the rest of the sequence suggested that the divergence between R1 and pSU316 replicons is very recent.

Fosfomycin-resistance plasmids determine an intracellular modification of fosfomycin

Escherichia coli cells carrying fosfomycin-resistance plasmids modify fosfomycin intracellularly. The product of this modification (fosfomycin-derivative) differs from fosfomycin in chromatographic mobility, but the chemical nature of the modification is not yet known. Fosfomycin-derivative appears to have a cytoplasmic location and lacks antibiotic activity. The modification system can be saturated by an excess of fosfomycin in the incubation media.

Foam cells from aorta and spleen overexpress apolipoprotein E in the absence of hypercholesterolemia

We have studied Apo E expression in atherosclerotic lesions and spleens in rabbits after a cholesterol-rich diet is discontinued and plasma cholesterol levels return to normal values. After 16 weeks, foam cells are still present in the atherosclerotic lesions and Apo E expression persists restricted solely to the lesion as ascertained by in situ RNA hybridization and northern blot. Apo E expression is induced in spleens of hypercholesterolemic rabbits. However Apo E mRNA levels decrease in this organ parallel to the disappearance of lipid loaded macrophages. These results of in vivo studies indicate that Apo E overexpression in foam cells does not depend on high serum cholesterol levels, but is a characteristic of macrophages that have acquired the foam cell phenotype.

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases.

Abstract Three mouse genes encoding type 1 protein phosphatase isotypes α, γ and δ were expressed in Schizosaccharomyces pombe cells under the control of the regulatable promoter nmt1. In the repressed state, basal expression of mouse genes was able to rescue the S. pombe mutant dis2-11. An integrated mouse gene could not, however, genetically complement S. pombe double mutants carrying disrupted dis2 and sds21 protein phosphatase genes. Overexpression of any of the three mouse protein phosphatase1 isotypes produced growth arrest and loss of viability in wild-type S. pombe cells. Overexpressing cells showed defects in chromosome distribution and in the formation of septa. These defects are characteristic of the expression of the mammalian protein phosphatases in S. pombe, since they are not observed after overexpression of endogeneous S. pombe type 1 protein phosphatases. Overexpression of a truncated version of isotype δ, which lacked protein phosphatase activity, reproduced most of the characteristics of the lethal phenotype. We conclude that the lethal effect may be due to interactions with essential cell cycle proteins.

Isolation and evolutionary analysis of a RepFVIB replicon of the plasmid pSU212

We have isolated at least two different replication regions from pSU401, a Tn802 insertion derivative of the IncFVI plasmid pSU212. One of the replication regions (RepFVIB) is highly homologous to the RepFIIA replicon of IncFII plasmids, and thus belongs to the RepFIIA family. We have also cloned the incompatibility determinant (incFVI) and the copy number control gene (cop) from RepFVIB and determined their nucleotide sequences. The analysis of the sequences supports the idea of a modular evolution of RepFIIA plasmids.

Hypercholesterolemia induces differential expression of rabbit apolipoprotein A and C genes

We have compared steady-state mRNA levels of apolipoproteins AI, AII, AIV, CI, CII and CIII in liver and small intestine of rabbits fed on a cholesterol-rich diet for up to 16 weeks. Apolipoprotein (apo) AIV mRNA was detected in both liver and small intestine, while apo AII was not detected in either organ. Apo CI, apo CII and apo CIII were expressed only in liver and apo AI mRNA was detected only in small intestine. In small intestine, apo AIV and apo AI mRNA levels increased to a maximum at the 4th and 12th week of treatment, respectively. In liver there was a parallel increase in the mRNA levels of apo AIV, apo CI, apo CII and apo CIII, with maximum levels after 4 weeks of treatment. A 3-fold increase was found in apo CII and apo CIII hepatic transcription rates between hypercholesterolemic and control rabbits after 4 weeks of treatment, no longer detectable after 8 weeks. However, no changes were found in apo AIV and apo CI transcription rates. Changes in apolipoprotein mRNA levels were accompanied by changes in plasma lipoprotein levels. Overall, these changes correlate well with the variations detected in the expression of the different apolipoprotein genes. Our results indicate that dietary cholesterol plays an important role in the regulation of these genes and that this regulation is tissue dependent.

Cloning and expression in minicells of the determinant of resistance to fosfomycin from the transposon Tn2921

The fosfomycin resistance determinant of the transposon Tn2921 has been localized and cloned into the plasmid pBR322. A DNA fragment of 1 kb contains all the information required for full expression of the resistance. The level of resistance was unaffected by the plasmid copy number and by the orientation in which the fragment was cloned. Studies in minicells showed that this 1-kb fragment directed the synthesis of a single polypeptide with a molecular mass of 16 kDa.