Defective colour vision associated with a missense mutation in the human green visual pigment gene.

All red/green colour vision defects described so far have been associated with gross rearrangements within the red/green opsin gene array (Xq28). We now describe a male with severe deuteranomaly without such a rearrangement. A substitution of a highly conserved cysteine by arginine at position 203 in the green opsins presumably accounted for his colour vision defect. Surprisingly, this mutation was fairly common (2%) in the population but apparently was not always expressed. In analogy with nonexpression of some 5'green-red hybrid genes in persons with normal colour vision, we suggest that failure of manifestation occurs when the mutant gene is located at a distal (3') position among several green opsin genes. This mutation might also predispose to certain X-linked retinal dystrophies.

Glucose-sensing mechanisms in eukaryotic cells.

Glucose not only serves as a nutrient but also exerts many hormone-like regulatory effects in a wide variety of eukaryotic cell types. Recently, interest in identifying general mechanisms and principles used to sense the presence of glucose has significantly increased and promising advances have been made: in yeast, the first proteins with an apparently specific function in glucose detection have been discovered; in plant cells, there is increasing evidence for a diverse array of glucose-induced signalling mechanisms; and in mammals, glucose-sensing phenomena have turned out to be much more widespread than just in the well-known example of pancreatic beta cells.

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin.

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.

An effect of androgens on the length of the poly(A)-tail and alternative splicing cause size heterogeneity of the messenger ribonucleic acids encoding cystatin-related protein.

The 22-kilodalton glycoprotein, expressed in the rat ventral prostate under the influence of androgens, is structurally a cystatin-related protein (CRP), as has been shown by copy DNA sequencing. In fact, two slightly different forms (CRP-1 and CRP-2) are expressed in the prostate; one of them (CRP-1) is also expressed in the exorbital lacrymal gland. In both glands, the CRP-1 messenger RNA (mRNA)s are androgen regulated. Moreover, androgens also influence the size of these mRNAs, which show marked heterogeneity (from 760-950 nucleotides). Indeed, the smaller forms are predominant in castrated animals, whereas the large forms are observed immediately after androgen induction. Hybridization with oligo(dT) followed by ribonuclease H treatment revealed that differences in length of the poly(A)-tail are responsible for this effect of androgens. Indeed, two well defined forms of CRP mRNA subsisted after removal of the poly(A)-tail by this treatment. In the less abundant shorter form (CRP-1 delta), 123 nucleotides are deleted by alternative splicing at the junction between the third and the fourth exon. The variant mRNA encodes a truncated protein, wherein the last 27 amino acids are replaced by a hydrophobic stretch of 8 amino acids. No alternative splicing was observed for the CRP-2 mRNA.

Binding of androgen-receptor complexes to alpha 2u-globulin genes and to the long terminal repeat of mouse mammary tumor virus.

The binding of androgen-receptor complexes to fragments derived from two alpha 2u-globulin genes (RAP 01 and RAO 01) was studied using a DNA-cellulose competition assay. Rat prostate cytosol labelled with [3H]mibolerone was used as a source of the androgen receptor. Two controls were included in these studies: the long terminal repeat (LTR) of mouse mammary tumor virus which has previously been shown to act as an androgen response element and a fragment of the C3 gene of prostatic binding protein which has been demonstrated to bind androgen-receptor complexes. Our experiments indicate that androgen-receptor complexes bind specifically and with comparable affinity to the C3 gene fragment, the LTR and a fragment of RAP 01 located in the 5'-upstream region (bp -642 up to -584). No specific interaction was observed with fragments derived from RAO 01. The region of RAP 01 which binds androgen-receptor complexes has previously been shown to interact with glucocorticoid receptors and contains a 17 bp sequence homologous with the consensus sequence for glucocorticoid-receptor binding. A mutation in this sequence in RAO 01 may be responsible for the loss of glucocorticoid and androgen-receptor binding. It is concluded that at least one member of the alpha 2u-globulin gene family interacts directly with androgen-receptor complexes with an affinity comparable to that observed for other androgen-dependent genes. The binding is observed in a region displaying also affinity for the glucocorticoid receptor.

Kallikrein-related protease in the rat ventral prostate: cDNA cloning and androgen regulation.

A kallikrein-related protease was purified from rat ventral prostate cytosol by means of DEAE-Sepharose chromatography, followed by gel filtration on Sephadex G-100 and CM-cellulose chromatography. Antibodies raised in rabbits against the purified protease recognize two bands on immunoblots of prostatic cytosol: a 31,000 Da band and an 18,000 Da band, which constitutes a proteolytic breakdown product of the former. The corresponding cDNA was isolated from a prostatic cDNA library, inserted in a lambda gt11 vector, using immunodetection for screening and identified as encoding a kallikrein- and tonin-related protease. Castration resulted in a marked decrease of the level of the protease and its mRNA, whereas administration of androgens to castrated animals resulted in marked stimulation. These data support the hypothesis that this protease is a member of a cluster of proteins, that are regulated in parallel by androgens in prostatic epithelial cells.

Androgen-dependent expression of cystatin-related protein (CRP) in the exorbital lacrimal gland of the rat.

Cystatin-related protein (CRP), also known as 20 (22)-kDa glycoprotein is expressed not only in the ventral prostate, but also in the lacrimal gland of adult male rats. In this study the expression of CRP in androgen-treated female animals is studied. CRP mRNA is absent in the lacrimal gland of untreated adult female rats, but can be induced by androgens, although this induction is slower than in castrated male rats. Estradiol, progesterone or glucocorticoids have no effect. In testicular feminized rats, however, CRP mRNA is not induced in the lacrimal gland by androgens. At the protein level, the presence of CRP in tears of adult male rats is demonstrated. In female animals or castrated male animals CRP can be induced by androgens in a dose-dependent way. Here also the induction is slower in female rats, even during secondary induction after previous full stimulation by androgens. These results indicate that androgens and a functionally normal androgen receptor are essential for the expression of CRP in the lacrimal gland. The time course of induction depends on the dose of androgens, the previous contact with androgens, the duration of the androgen-free interval and the sex of the animals.

Multiple binding sites for nuclear factors in the 5'-upstream region of two alpha 2u-globulin genes: implications for hormone-regulated and tissue-specific control.

In order to understand the tissue- and hormone-specific control of alpha 2u-globulin synthesis we isolated the 5'-upstream putative regulatory region of two alpha 2u-globulin genes: RAP 01 and RAO 01. Both clones seem to be expressed in rat liver. DNAseI footprinting analysis after incubation with rat liver nuclear extracts was used to identify regions of potential interest. Particular attention was paid to protected regions located in the neighbourhood of domains which, according to our previous studies, interact specifically with androgen- and glucocorticoid-receptor complexes. Fifteen DNAseI footprints could be mapped in clone RAO 01 (bp -758 up to the cap site). Nineteen footprints were observed in the corresponding region of RAP 01. Differences in the footprinting patterns were mainly observed in the more distal regions. Our data confirm the presence in both clones of two binding sites for the liver enriched factor pseudo-NF1 and one site for C/EBP previously observed in other alpha 2u-globulin genes. In addition we have been able to demonstrate, in RAP 01 only, a binding site for transferrin-liver factor 1. No differences in footprinting patterns could be demonstrated using liver nuclear extracts derived from animals with a high hepatic expression of alpha 2u-globulins (normal male rats) and animals with low to absent expression (prepubertal rats, female rats, rats with the testicular feminization syndrome, diabetic rats and hypophysectomized animals). Transfection experiments indicate that a fragment of RAP 01 (bp -643 up to -617) is able to act as a glucocorticoid and as an androgen response element. Larger fragments of RAP 01 and fragments of RAO 01 are ineffective. It is concluded that the expression of individual alpha 2u-globulin genes is probably the result of combinatorial interactions of several trans-acting factors with appropriate cis-acting elements. Moreover, important sites for tissue-specific and hormone-regulated expression may be situated outside the regions investigated.

Serine/alanine amino acid polymorphism of the L-cone photopigment assessed by dual Rayleigh-type color matches.

The dual Rayleigh-type color match is the ratio of 621 nm light to 550 nm light that in admixture matches 586 nm light, divided by the ratio of 667 nm light to 550 nm light that in admixture matches 586 nm light. Compared to the classical Rayleigh match, the dual-match procedure minimizes variation in color matching arising from differences in lens pigmentation and photopigment optical density, and thus amplifies individual differences due to shifts in L pigment lambda max. We hypothesized that the dual matches would provide a clearer distinction between subjects with serine and subjects with alanine than would the classical Rayleigh match because individuals with serine express L pigments with a lambda max shifted toward longer wavelengths than do those with alanine. Classical Rayleigh color matches were compared with dual Rayleigh-type color matches in 14 color-normal observers whose DNA had been analyzed previously for the presence of the amino acid serine or alanine at position 180 in the L opsin. The resulting distribution of dual-match measurements for the seven subjects with serine does not overlap the distribution of measurements for the seven subjects with alanine. The classical Rayleigh-match measurements for these two groups of subjects, on the other hand, overlap substantially. More than half of the subjects' classical Rayleigh matches are within the overlapping range. The dual Rayleigh-type matches, therefore, provide an improved psychophysical technique for assessing whether an individual observer has serine or alanine at position 180.

Serine/alanine amino acid polymorphism of the L and M cone pigments: effects on Rayleigh matches among deuteranopes, protanopes and color normal observers.

In a first experiment, groups of deuteranopes and protanopes were characterized psychophysically by the slopes of regression lines fitted to yellow intensity settings from their Rayleigh matches. In a second experiment, color normal male subjects were characterized by their 2 and 8 deg Rayleigh match points. All subjects had been previously characterized genetically by the presence of the amino acid serine or alanine at position 180 on their L cone or L/M hybrid opsins. Dichromats and color normal subjects with serine had greater sensitivity to the red primary than did those with alanine. Calculations based on psychophysical results suggest that the substitution of serine by alanine in the L cone opsin or L/M hybrid opsin produces a shift in lambda max of the expressed pigment toward shorter wavelengths by an amount varying between 2.6 and 4.3 nm, with the shifts in lambda max for the dichromats being larger than those for the color normal subjects.

A specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K176M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, proliferation and stress resistance. Derepressed cells show a rapid increase in the cAMP level (within 1 min) after addition of glucose or after intracellular acidification. A specific mutation in adenylate cyclase, the enzyme that catalyzes the synthesis in cAMP, largely prevents both cAMP responses. The responsible mutation was originally called lcr1 (for lack of cAMP responses); lcr1 was later identified as allelic with CYR1/CDC35. The mutation was introduced into the CYR1 gene of a W303-1A wild type strain, which resulted in a large decrease in cAMP signalling. Furthermore, there was a strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated adenylate cyclase activity in isolated plasma membranes, which is consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the lcr1 mutant. Because of the absence of cAMP signalling during exponential growth on glucose, it was concluded that glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Activation of the PKA pathway is mediated by a G protein (either Ras1/Ras2 or Gpa2). Constitutive activation of the pathway by Ras2val19 or Gpa2val132 has a negative effect on glycogen and trehalose accumulation and heat shock survival. The lcr1 mutation partially suppresses this effect indicating that the target sites of the two G-proteins on adenylate cyclase might have at least a part in common.

The benefits of humanized yeast models to study Parkinson's disease.

Over the past decade, the baker's yeast Saccharomyces cerevisiae has proven to be a useful model system to investigate fundamental questions concerning the pathogenic role of human proteins in neurodegenerative diseases such as Parkinson's disease (PD). These so-called humanized yeast models for PD initially focused on α -synuclein, which plays a key role in the etiology of PD. Upon expression of this human protein in the baker's yeast Saccharomyces cerevisiae, the events leading to aggregation and the molecular mechanisms that result in cellular toxicity are faithfully reproduced. More recently, a similar model to study the presumed pathobiology of the α -synuclein interaction partner synphilin-1 has been established. In this review we will discuss recent advances using these humanized yeast models, pointing to new roles for cell wall integrity signaling, Ca(2+) homeostasis, mitophagy, and the cytoskeleton.

The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to α-synuclein toxicity in Parkinson's disease models.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Yeast unfolds the road map toward alpha-synuclein-induced cell death.

The budding yeast Saccharomyces cerevisiae has contributed significantly to our current understanding of eukaryotic cell biology. It served as a tool and model for unraveling the molecular basis of a wide variety of cellular phenomena, which seem to be conserved in other organisms. During the last decade, yeast has also extensively been used to study the mechanisms underlying several human diseases, including age-associated neurodegenerative disorders, such as Parkinson's, Huntington's and Alzheimer's disease. In this review, we focus on a yeast model for synucleinopathies and summarize recent studies that not only provided new clues on how the misfolding of alpha-synuclein (alpha-syn) triggers toxicity and eventually cell death, but that also led to the identification of conserved suppressor proteins, which are effective in protecting cells, including neurons, from the alpha-syn-induced cytotoxicity.

Ydc1p ceramidase triggers organelle fragmentation, apoptosis and accelerated ageing in yeast.

Saccharomyces cerevisiae dihydroceramidase Ydc1p hydrolyzes ceramide, resulting in accumulation of free long-chain bases and their phosphates. Yeast mutants lacking YDC1 are characterized by increased chronological lifespan. Moreover, we found YDC1 up-regulated in a yeast mutant displaying reduced chronological lifespan. These data suggest an important role for Ydc1p in chronological lifespan determination in yeast. Mitochondria are known to play an important role in chronological lifespan and apoptosis. In this study we demonstrated that overexpression of YDC1 results in reduced chronological lifespan and increased apoptotic cell death. We found YDC1 overexpression to result in mitochondrial fragmentation and dysfunction. Interestingly, vacuoles also appeared to be fragmented and dysfunctional upon YDC1 overexpressing. Exogenous addition of ceramide to YDC1-overexpressing cultures increased chronological lifespan and restored organelle function. In conclusion, this study describes a direct link between ceramide metabolism in yeast and mitochondrial and vacuolar fragmentation and function, with consequences for chronological lifespan in yeast.

Haplotype diversity in the human red and green opsin genes: evidence for frequent sequence exchange in exon 3.

We studied polymorphisms in the coding sequences of the human red and green opsin genes of 133 Caucasian males. Eleven polymorphic sites were discovered in the red opsin gene, seven of which were in exon 3, three in exon 4 and one in exon 5. Polymorphisms at 8 of these sites resulted in amino acid substitutions which generated a total of 18 unique red opsins in the population. The substitutions at three (S180A, I230T, and A233S) of the 8 sites involve hydroxyl-bearing to non-polar amino acid residues, and are therefore likely to alter spectral characteristics of the red pigment. Eight polymorphic sites were observed in the green opsin coding sequences, six of which were in exon 3, one in exon 2 and one in exon 5. Five of the eight involved amino acid substitutions which generated 15 unique green opsins in the population. Substitutions at two of these sites involve hydroxyl-bearing vs. non-polar residues. Six polymorphisms, all of which are located in exon 3, are shared between the red and green opsin genes, essentially making it difficult to assign this exon to either of these genes. Markers in exon 3 are in partial linkage disequilibrium with those in exons 4 and 5, whereas the latter two are in strong linkage disequilibrium with each other. Furthermore, markers in the 5' region of exon 3 are also in only partial (54%) disequilibrium with those in the 3' region. The above results strongly suggest a history of frequent gene conversion, mainly localized to exon 3, in the lineages leading to the human red and green opsin genes.(ABSTRACT TRUNCATED AT 250 WORDS)