Biomedical subjects
Publications and source records attributed to R P Jansen.
Cytoplasmic messenger RNA localization is a key post-transcriptional mechanism of establishing spatially restricted protein synthesis. The characterization of cis-acting signals within localized mRNAs, and the identification of trans-acting factors that recognize these signals, has opened avenues towards identifying the machinery and mechanisms involved in mRNA transport and localization.
Germline mutations in either of the two major breast cancer predisposition genes, BRCA1 and BRCA2, account for a significant proportion of hereditary breast/ovarian cancer. Identification of breast cancer patients carrying mutations of these genes is primarily based on a positive family history of breast/ovarian cancer or early onset of the disease or both. In the course of mutation screening of the BRCA1 and BRCA2 genes in a hospital based series of patients with risk factors for hereditary breast/ovarian cancer, we identified a germline mutation in the BRCA2 gene (3034del4) in a patient with early onset breast cancer and no strong family history of the disease. Subsequent molecular analysis in her parents showed that neither of them carried the mutation. Paternity was confirmed using a set of highly polymorphic markers, showing that the proband carried a de novo germline mutation in the BRCA2 gene. Interestingly, 3034del4 is a recurrent mutation occurring in a putative mutation prone region of the BRCA2 gene. Our study presents the first case in which a de novo germline mutation in the BRCA2 gene has been identified, and supports previous results of haplotype studies, confirming that the 3034del4 mutation has multiple independent origins.
RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences ('zipcodes') within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p's C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA-motor protein adapters.
The mitochondrial genome comprises a circular, histone-free 'chromosome' of 16.6 kb of DNA, present in one or more copies in every mitochondrion. This chromosome has been tightly conserved for more than half a billion years, coding in every multicellular animal so far investigated, both vertebrate and invertebrate: (i) the same 13 protein subunits required for oxidative phosphorylation; (ii) a component of each of the two mitochondrial ribosome subunits; and (iii) the 22 transport RNAs present within the mitochondrion. Exons on the circle are tightly packed, with no spacing introns. Mitochondrial DNA is histone-free, has limited repair ability, and has a relatively high mutation-fixation rate. Inheritance is cytoplasmic and maternal, with epidemiological evidence (namely the familial distribution of polymorphisms) indicating that recombination with mtDNA of paternal origin is exceedingly rare. Thus the maintenance and evolution of mtDNA (its remarkably successful symbiotic persistence with the nuclear genome) has been essentially asexual. The machinery for homologous recombination is present in mitochondria of at least some species, however, and it might be surprising if it did not occur between circles in some circumstances. By bringing together the fields of mitochondrial biochemistry, evolutionary genetics, reproductive physiology, and neuromuscular medicine in focusing on the inheritance of normal and abnormal human mtDNA, we can hope to better understand the forces behind this genome's inheritance and what might be required of ovarian function to satisfy its accurate persistence over millions of years. Clinically we can hope also for a better understanding of ooplasmic factors in human fertility and in the wide manifestations of mitochondrial genomic disease.
Using a semi-quantitative review of published electron micrographs, we have explored the passage of mitochondria from one generation to the next through the cytoplasm of the human female germ cell. We propose a testable hypothesis that the mitochondria of the germline are persistently 'haploid' (effectively carrying just one mitochondrial chromosome per organelle). For mitochondria, the passage through germ cell differentiation, oogenesis, follicle formation and loss could constitute a restriction/amplification/constraint event of a type previously demonstrated for asexual purification and refinement of a nonrecombining genome. At the restriction event (or 'bottleneck') in the human primordial germ cell, which differentiates in embryos after gastrulation, there appear to be <10 mitochondria per cell. From approximately 100 or so such cells, a population of > or =7 x 10(6) oogonia and primary oocytes is produced in the fetal ovaries during mid-gestation, with mitochondria numbering up to 10000 per cell, implying a massive amplification of the mitochondrial genome. A further 10-fold or greater increase in mitochondrial numbers per oocyte occurs during adult follicular growth and development, as resting primordial follicles develop to preovulatory maturity. So few are the numbers of oocytes that fertilize and successfully cleave to form an embryo of the new generation, that biologists have long suspected that a competitive constraint lies behind the generational completion of this genetic cycle. I propose that maintaining the integrity of mitochondrial inheritance is such a strong evolutionary imperative that features of ovarian follicular formation, function, and loss could be expected to have been primarily adapted to this special purpose. To extend the hypothesis further, the imperative of maintaining mitochondrial genomic integrity in a population could explain why women normally become sterile a number of years before there is depletion of ovarian follicles and endocrine ovarian failure (i.e. why there is 'an oopause' preceding the menopause). Plausible explanations might also follow for several well-known and puzzling reproductive difficulties, including recurrent miscarriage, unexplained infertility, and persistent failure of IVF embryos to cleave or to implant. Current experimental laboratory manoeuvres that might circumvent mitochondrial shortcomings (such as cytoplasmic transfusion and karyoplast exchange) are examined and possible clinical hazards identified.
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Asymmetric distribution of messenger RNAs is a widespread mechanism to localize synthesis of specific protein to distinct sites in the cell. Although not proven yet there is considerable evidence that mRNA localisation is an active process that depends on the activity of cytoskeletal motor proteins. To date, the only motor protein with a specific role in mRNA localisation is the budding yeast type V myosin Myo4p. Myo4p is required for the localisation of ASH1 mRNA, encoding a transcriptional repressor that is essential for differential expression of the HO gene and mating type switching in budding yeast. Mutations in Myo4p, in proteins of the actin cytoskeleton, and in four other specific genes, SHE2-SHE5 disrupt the daughter-specific localisation of ASH1 mRNA. In order to understand if Myo4p is directly participating in mRNA transport, we used in situ colocalisation and coprecipitation of Myo4p and ASH1 mRNA to test for their interaction. Our results indicate an association of Myo4p and ASH1 mRNA that depends on the activity of two other genes involved in ASH1 mRNA localisation, SHE2 and SHE3. This strongly suggests a direct role of Myo4p myosin as a transporter of localised mRNAs, convincingly supporting the concept of motor-protein based mRNA localisation.
It has become evident over the past years that a large fraction of messenger RNAs is tightly associated with the cytoskeleton. Whereas microtubules are involved in RNA-cytoskeletal association in large cells like oocytes, neurons, or oligodendrocytes, microfilaments play the major role in smaller somatic cell types. Association of RNA with cytoskeletal filaments clearly is required for mRNA transport, but also appears to be crucial for efficient protein synthesis. Recent data now shed light on how mRNAs attach to the cytoskeleton. Messenger RNA seems to interact with microtubules or microfilaments in the form of large ribonucleoprotein particles, which in some cases also contain components of the protein synthesis apparatus. Recently, a number of RNA binding proteins have been identified in flies, amphibians, and mammals that are essential for the interaction of mRNA with cytoskeletal filaments or with microtubule- or actin-associated proteins. Such proteins include heterologous ribonucleoproteins, which are also involved in nuclear export of RNA.
Molecular geneticists and ovarian physiologists today face the challenge of defining and reconciling two major biological imperatives that each center on oogenesis, folliculogenesis and competition between ovarian follicles: (1), defining how the mitochondrial genome--important in both aging and a number of serious mitochondrial diseases--is refreshed and purified as it passes, via the oocyte's cytoplasm, from one generation to the next; and (2), endeavouring to discover what cytoplasmic factor(s) it is that permits some eggs but not others to produce viable embryos and ongoing pregnancies. We review here in detail the passage of mitochondria through the female germ cell line. For mitochondria, the processes of oogenesis, follicle formation and loss constitute a restriction/amplification/constraint event of the kind predicted by L. Chao for purification and refinement of a haploid genome. We argue that maintaining the integrity of mitochondrial inheritance is such a strong evolutionary imperative that we should expect at least some features of ovarian follicular formation, function and loss to be primarily adapted to this specific purpose. We predict, moreover, that to prevent accumulation of mild mitochondrial genomes in the population there is a need for physiological female sterility prior to total depletion of ovarian oocytes, a phenomenon for which there is empirical evidence and which we term the oöpause.
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Cell divisions that produce progeny differing in their patterns of gene expression are key to the development of multicellular organisms. In the budding yeast Saccharomyces cerevisiae, mother cells but not daughter cells can switch mating type because they selectively express the HO endonuclease gene. This asymmetry is due to the preferential accumulation of an unstable transcriptional repressor protein, Ash1p, in daughter cell nuclei. Here it is shown that ASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin. A cis-acting element in the 3'-untranslated region of ASH1 mRNA is sufficient to localize a chimeric RNA to daughter cells. These results suggest that localization of mRNA may have been an early property of the eukaryotic lineage.
Asymmetric distribution of cytoplasmic proteins and messenger RNAs has been implicated in several instances of cell differentiation. Microtubules have been suggested to direct mRNA localization in Drosophila and Xenopus oocytes but motor proteins that might transport mRNAs have not yet been identified. Recent data imply that in Drosophila, Caenorhabditis elegans and budding yeast, proteins of the actin cytoskeleton, including unconventional myosins, play active roles in the segregation of differentiation factors and mRNAs.
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Certain cell types give rise to progeny that adopt different patterns of gene expression in the absence of any differences in their environment. Cells of budding yeast give birth to mother and daughter cells that differ in that only mother cells express the HO endonuclease gene and thereby switch mating types. We describe the identification of five genes, called SHE1-SHE5, that encode cytoplasmic proteins required for mother-specific HO expression. She1p, which is identical to the minimyosin Myo4p, and She3p are not, however, mother-specific proteins. On the contrary, they accumulate in growing buds. She proteins might be required for the transport of factors that promote HO repression from the mother cell into its bud. In an accompanying paper, we show that SHE genes are needed for the accumulation in daughter nuclei of Ash1p, a repressor of HO.
Cell division in haploid yeast gives rise to a "mother" cell capable of mating-type switching and a "daughter" cell that is not. Switching is initiated by the HO endonuclease, whose gene is only transcribed in cells that have previously given birth to a bud (mother cells). HO expression depends on a minimyosin, She1p/Myo4p, which accumulates preferentially in growing buds. We describe a gene, ASH1, that is necessary to repress HO in daughters. ASH1 encodes a zinc finger protein whose preferential accumulation in daughter cell nuclei at the end of anaphase depends on She1p/Myo4p. The greater abundance of Ash1p in daughter cells is responsible for restricting HO expression to mother cells.
Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.
We describe two female monozygotic (MZ) twins heterozygous for Fabry disease, an X linked disorder resulting from the deficient activity of alpha-galactosidase A. While one of the twins was clinically affected, the other was asymptomatic. Enzymatic assay of alpha-galactosidase in blood leucocytes, skin fibroblasts, Epstein-Barr virus transformed lymphoid cell lines, and hair follicles of the twins and their parents confirmed the heterozygous status of the twins and indicated that Fabry disease had occurred as a result of a de novo mutation. The son of the unaffected twin sister was shown to be hemizygous. Molecular analysis of the alpha-galactosidase A gene permitted the identification of an as yet undescribed point mutation at position 10182 of exon 5 which causes an Asp to Asn substitution at codon 231. Single strand conformation polymorphism (SSCP) analysis again showed the heterozygous status of the twins and a normal pattern in their parents. The basis for the discordant expression of this d novo mutation in the twins was investigated by studying their X inactivation status. Analysis of the inactive X specific methylation at the androgen receptor gene showed unbalanced inactivation in the twins' fibroblasts and in opposite directions. While the maternally derived X chromosome was preferentially active in the asymptomatic twin, the paternal X chromosome was active in the other, affected twin and was found in her hemizygotic nephew. These data suggest that the paternal X chromosome carries the de novo alpha-galactosidase A mutation and that uneven X inactivation is the underlying mechanism for disease expression in this novel female MZ twin pair. This is the first documented case of female twins discordant for Fabry disease.
The mucous surfaces of the oviducts of mammals, especially humans, are dependent on estradiol. The mucus glycoproteins and glycocalyceal glycoproteins have, however, barely been studied. Biochemical analyses have focussed on the relatively low molecular weight glycoproteins likely to be found in more serous-type granules of the ampulla and not on the very high MW glycoproteins typical of mucus and represented in the isthmus by morphological evidence of mucus secretion. Quantitatively, secretion from the ampulla is likely to predominate, because of its huge surface area compared with the isthmus. But functional closure of the isthmus under the influence of estradiol in the absence of progesterone means that it is the isthmus where luminal secretions accumulate--and where mucus glycoproteins will exert their most important effects on spermatozoa ascending the reproductive tract, and then on fertilized ova en route to the uterus. Further study of the extracellular, intraluminal, carbohydrate-rich environment of the oviductal isthmus, especially in humans, is likely to prove rewarding. Sampling of these secretions is now feasible using transvaginal, transuterine fallopian tube catheters that are in clinical use.