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Biomedical subjects

H van der Spek

Publications and source records attributed to H van der Spek.

12 recordsLinked to original sources
PubMed2006

The impact of functional genomics on microbiological food quality and safety.

In the food processing industry, unwanted occurrence and growth of spoilage and pathogenic microorganisms is a key concern. A prime example is the extremely heat resistant bacterial endospores, microbial survival structures, that create problems due to their ability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. Research on food spoilage Bacillus subtilis isolates using the Amplified Fragment Length Polymorphism (AFLP) technology and micro-array technology has identified a number of genomic factors correlated to the level of spore heat resistance. Strains could be classified according to these genomic markers. In addition, it was shown with the sequenced B. subtilis laboratory strain that sporulation in the presence of in particular calcium ions in a cocktail of calcium, magnesium, iron, manganese and potassium promotes thermal resistance of developing spores. This physiological observation correlated with an increased expression during sporulation of genes encoding small acid soluble spore proteins. Screening of ingredients using DNA-chip based techniques identifying the above indicated molecular markers, should allow in the future the identification of the occurrence of spoilage and pathogenic bacteria and prediction of their thermal preservation stress resistance. Currently various projects aiming at the integration of genomics data and micro(nano)-technology, a prerequisite if the alluded to ingredient Quality Control is going to succeed, are running or are being set-up. Information from these projects will be used together with the requirements of product organoleptic quality to derive robust integrated food safety and food quality processing parameters. Such parameters should form the basis of future food Quality Assurance systems.

Bacillus subtilis↗
PubMed2000

Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD(+))-dependent isocitrate dehydrogenase.

We have previously demonstrated that the yeast Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5'-untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2-3-fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2-3-fold reduced half-life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs.

Adenosine Triphosphatases↗
PubMed2000

The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant.

In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting. When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37 degrees C. We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.

ATP-Dependent Proteases↗
PubMed2000

Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins.

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

Amino Acid Sequence↗
PubMed2000

Isolation and RNA-binding analysis of NAD+ -isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe.

Krebs cycle NAD+ -isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiae COX2 leaders. In contrast. Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA.

Amino Acid Sequence↗
PubMed2000

Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae that lack a functional MSS51 gene are respiratory deficient due to the absence of cytochrome c oxidase subunit 1 (Cox1p). It has been previously suggested, but not formally proven, that Mss51p is required for translational activation of COX1 mRNA, rather than being involved in a subsequent step in the synthesis of Cox1p or its assembly into cytochrome c oxidase. Pulse-chase labelling experiments now show that the absence of detectable levels of Cox1p in mss51-null strains is indeed due to the lack of synthesis of Cox1p, and is not caused by reduced stability of the protein. To gain more insight into the exact function of Mss51p, we determined the subcellular localization of the protein. We were able to show that an epitope-tagged version of Mss51p (Mss51HA) complements the mutation and can be localized in mitochondria, where it is firmly associated with the mitochondrial inner membrane. In addition, we characterized the previously identified mutant allele mss51-3. Sequence analysis revealed the presence of a short open reading frame upstream of MSS51 resulting from the creation of an extra ATG start-codon.

Alleles↗
PubMed1999

Mitochondrial assembly in yeast.

The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up. A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelle's own genetic system, which itself contributes seven key components of the electron transport chain. Translation and respiratory complex assembly are particularly expensive processes, together requiring around 150 of the proteins so far known. Recent developments in both areas are reviewed and approaches to the identification of novel mitochondrial proteins are discussed.

Animals↗
PubMed1994

RNA editing in mitochondria of cultured trypanosomatids: translatable mRNAs for NADH-dehydrogenase subunits are missing.

RNA editing in mitochondria of kinetoplastid protozoa involves the posttranscriptional insertion and deletion of uridylate residues in protein encoding regions of pre-mRNAs. Editing is required to remove gene-encoded translational defects or to convert a nonsense sequence into a sense message. In cultured trypanosomatids, however, translationally defective pre-mRNAs for a number of NADH-dehydrogenase subunits are not converted into functional mRNAs by editing. In this report, the available data are discussed in the context of current models for RNA editing.

Animals↗
PubMed1993

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.

Animals↗
PubMed1991

Conserved genes encode guide RNAs in mitochondria of Crithidia fasciculata.

RNA editing is the post-transcriptional alteration of the nucleotide sequence of RNA, which in trypanosome mitochondria is characterized by the insertion and deletion of uridine residues. It has recently been proposed that the information for the sequence alteration in Leishmania tarentolae is provided by small guide (g) RNAs encoded in the mitochondrial DNA [Blum et al. (1990) Cell, 60, 189-198]. We are studying the mechanism of RNA editing in the insect trypanosome Crithidia fasciculata and report that: (i) a full length, conventional DNA gene or an independently replicating RNA gene that could encode the edited MURF3 transcript is absent when probed for in sensitive, calibrated assay systems; (ii) in all cases (seven) investigated in C. fasciculata so far, putative gRNA genes are found in a position in the mitochondrial DNA virtually identical to that in L. tarentolae and (iii) also in C. fasciculata, the putative gRNA genes are transcribed into small RNAs with discrete 5' ends. These results provide strong evolutionary evidence in support of the participation of gRNAs in RNA editing. Remarkably, in C. fasciculata the basepaired region of some putative gRNA:mRNA hybrids contains a C:A non-Watson-Crick basepair.

Animals↗
PubMed1988

Transcripts from the frameshifted MURF3 gene from Crithidia fasciculata are edited by U insertion at multiple sites.

In trypanosome mitochondria an RNA editing process is operative, which co- or post-transcriptionally alters the nucleotide sequence of transcripts by insertion and/or deletion of U residues at specific sites. To increase our understanding of the mechanism of this process we have compared the nucleotide sequence of the frameshifted mitochondrial MURF3 gene from Crithidia fasciculata to that of a large number of MURF3 cDNAs. We found cDNAs derived from transcripts edited at two different sites in the protein coding sequence: (i) at the frameshift position five extra U residues connect the two reading frames and (ii) at the 5' terminus 22 inserted Us shift a putative initiator codon out of phase. The collection also contained cDNAs that were derived from non-edited transcripts. Partially edited sequences were not found, except in one cDNA, which contained an edited frameshift site in combination with a non-edited 5' terminus. The analysis further showed that MURF3 transcripts have a 3'-terminal poly(AU) extension, which varies in sequence. The implications of these results are discussed.

Amino Acid Sequence↗