OBJECTIVE

This article reviews recent experimental advances in the development of biosynthetic implants for repair of spinal cord injury.

RESULTS

Various important advances in the development of biosynthetic conduits for spinal cord repair have recently been reported. It was found that implantation of freeze dried alginate sponge into completely transected spinal cord supports axonal regeneration across the lesion site. A poly(lactic-co-glycolic acid) scaffold seeded with neural stem cells has been developed that promotes axonal regeneration across the gap. It was found that polyethylene glycol can reseal damaged spinal cord axons and restore impulse conduction. Findings have been reported that poly-beta-hydroxybutyrate conduits in combination with alginate and fibronectin provide neuroprotection for axotomized descending neurones. It has been reported that conduits made of fibronectin mats or fibrin in combination with neurotrophic growth factors promote axonal growth into the grafts. Finally, magnetic resonance imaging after experimental spinal cord injury has been used to monitor regeneration in biosynthetic conduits in vivo over time.

CONCLUSIONS

Biosynthetic conduits carrying extracellular matrix molecules and different cell lines, and supplemented with neurotrophic growth factors have yielded encouraging results in the treatment of experimental spinal cord injury. These findings provide a basis for further development of techniques aimed at spinal cord repair in humans.

A new gene, STE50, which plays an essential role in cell differentiation in Saccharomyces cerevisiae was detected and analysed. STE50 expression is not cell type-specific and its expression in MATa and MAT alpha cells is unaffected by pheromones. When present on a high copy number plasmid, STE50 causes supersensitivity to alpha-pheromone, and increases the level of alpha-pheromone-induced transcription of FUS1 in haploid a cells. Mutants bearing either of the two gene disruptions, ste50-1 or ste50-2, are sterile and have a modulated sensitivity to alpha-pheromone. The overexpression of STE4 (G beta) in wild-type cells elicits a constitutive growth arrest signal, however this phenotype is suppressed by a C-terminal truncation mutation in STE50 (ste50-2). In contrast, the constitutive activation of the pheromone response pathway caused by disruption of GPA1 (G alpha) is not suppressed in ste50-2 mutants. The ste50-2 mutation partially suppresses the desensitisation defect of the sst2-1 mutation, and the resulting ste50-2 sst2-1 mutants restore fertility. Our results indicate that the ste50-2 mutant may have a defect in adaptation (hyperadaptation), and suggest a possible interaction of STE50-2 with the G alpha subunit of the G protein.

A novel oxygen microelectrode with a tip diameter of 2 to 20 mum was constructed which could function satisfactorily under a variety of environmental conditions and in a variety of media, including human blood serum, citric acid at pH 2.5, moist air, and paraffin oil. Measurement of oxygen by this electrode does not require stirring of the medium. Electrodes could be made so that the 90% response time necessary to detect changes in oxygen concentration was less than 0.2 s, and response was linear with oxygen concentration. To demonstrate the performance of the electrode, oxygen and photosynthesis profiles of an acid microbial mat (pH 2.8) dominated by the eucaryotic alga Cyanidium caldarium were made. Photosynthetic rates as high as 95 mmol of O(2) dm h were measured within the most active 0.1-mm layer, which was ca. 0.2 mm below the surface of the microbial mat. The total photosynthetic activity was 47 mmol of O(2) m h. Vertical profiles of photosynthesis at different light intensities showed that the microalgae within the mat were not photoinhibited at bright sunlight (2,090 muEinsteins m s).

Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Change of bacterial community occurring along a hot water stream in the Hishikari gold mine, Japan, was investigated by applying a combination of various culture-independent techniques. The stream, which is derived from a subsurface anaerobic aquifer containing plentiful CO2, CH4, H2, and NH4+, emerges in a mine tunnel 320 m below the surface providing nutrients for a lush microbial community that extends to a distance of approximately 7 m in the absence of sunlight-irradiation. Over this distance, the temperature decreases from 69 degrees C to 55 degrees C, and the oxidation-reduction potential increases from -130 mV to +59 mV. In the hot upper reaches of the stream, the dominant phylotypes were: 1) a deeply branching lineage of thermophilic methane-oxidizing gamma-Proteobacteria, and 2) a thermophilic hydrogen- and sulfur-oxidizing Sulfurihydrogenibium sp. In contrast, the prevailing phylotypes in the middle and lower parts of the stream were closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira spp.. Changes in the microbial metabolic potential estimated by competitive PCR analysis of genes encoding the enzymes, particulate methane monooxygenase (pmoA), ammonia monooxygenase (amoA), and putative nitrite oxidoreductase (norB), also substantiated the community shift indicated by 16S rRNA gene analysis. The diversity of putative norB lineages was assessed for the first time in the hot water environment. Estimation of dominant phylotypes by whole-cell fluorescent in situ hybridization and changes in inorganic nitrogen compounds such as decreasing ammonium and increasing nitrite and nitrate in the mat-interstitial water along the stream were consistent with the observed transition of the bacterial community structure in the stream.

Polymerase chain reaction assay (PCR) amplifying a fragment of the Leptospira rrs gene was compared with culture and microagglutination test (MAT) for the diagnosis of leptospirosis in a study of 200 patients with various clinical syndromes compatible with leptospirosis. For the first group of samples tested, PCR identified the 14 cases that later were unequivocally confirmed to be leptospirosis. Thirteen other systemic cases presenting decreasing leptospiral antibody titers were also detected by PCR. The average persistence of leptospiral DNA in serum was estimated at 12 days, with a maximum of 56 days in a culture-confirmed case. The possibility of detecting leptospires in aqueous humor during the ocular complications of the disease was confirmed. The results suggest that PCR is an efficient tool for early diagnosis of leptospirosis during the first 10 days of the disease, especially when the clinical expression of the disease is confusing.

A pluri-disciplinary in situ colonization experiment was performed to study early stages of colonization in deep-sea vent Alvinella spp. worm habitats. Four colonization devices were deployed onto Alvinella spp. colonies of different chimneys of the East-Pacific Rise (EPR 13 degrees N), for two different periods: a short (less than a week) and a longer one (3 weeks). Video imagery and monitoring of the thermal and physico-chemical conditions were performed during the colonization experiments. Numerous microorganisms bearing specialized adhesion-appendages and/or high amounts of polymeric extracellular matrix were observed on devices, which may efficiently contribute to the colonization of new surfaces. The microbial cohorts preceding and accompanying Alvinella spp. settlement were identified. In all cases, Archaea could not be detected and the microbial mats were essentially composed of e-Proteobacteria. Within this group, one phylotype (AlviH2) was found to dominate the libraries of three colonization devices. Dominance of e-Proteobacteria in the libraries may reflect the wide physiological variety encountered within this group or an adaptability of these microorganisms towards their changing environment. Bacteria affiliated to the Cytophaga-Flavobacterium-Bacteroides group or to the e-Proteobacteria, that grow either chemo-organoheterotrophically by fermentation or chemolithoautotrophically with H2 as an electron donor and S degrees /S2O32- or NO3- as a terminal electron acceptor, were isolated from one of the microbial mat formed in 20 days.

The aim of this study was to develop novel biomedicated nanofiber electrospun mats for controlled drug release, especially drug release directly to an injury site to accelerate wound healing. Nanofibers of poly(vinyl alcohol) (PVA), poly(vinyl acetate) (PVAc), and a 50:50 composite blend, loaded with ciprofloxacin HCl (CipHCl), were successfully prepared by an electrospinning technique for the first time. The morphology and average diameter of the electrospun nanofibers were investigated by scanning electron microscopy. X-ray diffraction studies indicated an amorphous distribution of the drug inside the nanofiber blend. Introducing the drug into polymeric solutions significantly decreased solution viscosities as well as nanofiber diameter. In vitro drug release evaluations showed that both the kind of polymer and the amount of drug loaded greatly affected the degree of swelling, weight loss, and initial burst and rate of drug release. Blending PVA and PVAc exhibited a useful and convenient method for electrospinning in order to control the rate and period of drug release in wound healing applications. Also, the thickness of the blend nanofiber mats strongly influenced the initial release and rate of drug release.

The Cabo Rojo Solar Salterns located on the southwest coast of Puerto Rico are composed of two main ecosystems (i.e., salt ponds and microbial mats). Even though these locations are characterized by high solar radiation (mean light intensity of 39 mol photons m-2d-1) they harbour a diverse microscopic life. We used morphological and molecular techniques to identify a series of halotolerant fungi. A total of 183 isolates and 36 species were cultured in this study. From the water from the salt ponds, 86 isolates of 26 species were cultured. The halotolerant fungi isolated from water were: Cladosporium cladosporioides, nine Aspergillus sp., five Penicillium sp. and the black yeast Hortaea werneckii. A distinctive isolate with a blue mycelium was cultured from the salt ponds, representing a new species of Periconia based on morphology and rDNA analysis. Forty-four isolates from eight species were cultured from the sediments around the salt ponds. Most of the sediment isolates formed only sterile mycelium, while several were Chaetomium globosum. A total of 53 isolates from 16 species were isolated from the three layers of the microbial mats, of which Aspergillus niger was the most frequent isolate. Phospholipid fatty acid profiles generated from the different layers of the microbial mats indicated that the uppermost layers of the mats contained fungal biomarker, 18:2w6. This fatty acid decreased with depth, the highest concentration was observed in the green upper layer and it disappeared in the black bottom anoxic layer. This correlates with the isolation of fungi using the serial dilution technique. This is the first study that documents the presence of fungi in microbial mats.

Cloning of cDNA obtained from 16S rRNA (16S rcDNA) selectively retrieves species-specific sequence information useful for analyzing the composition and structure of natural microbial communities. With this technique we obtained recombinant 16S rcDNA libraries from Escherichia coli and from a model hot-spring cyanobacterial-mat community. The recombinant plasmids contained exclusively 16S rRNA-derived inserts. This selective approach is independent of biasing culture techniques and eliminates the laborious screening required to locate 16S rRNA gene-bearing recombinants in genomic DNA libraries obtained from natural communities.

The aim of the present study was to characterize the performance ability of the leg extensor apparatus in a group of athletes with jumper's knee and to compare the results with those of a matched control group without knee symptoms. Patient and control groups (12 players in each) were selected from a population of 141 well-trained male Norwegian volleyball players, of which 55 (39%) satisfied the diagnostic criteria for jumper's knee. The testing program consisted of a standing jump, a countermovement jump, a 15-second rebound jump test, a standing jump with a 20-kg load, and a standing jump with a load corresponding to one-half of the subject's body weight. Jump height and power were measured using a contact mat connected to an electronic timer. The test results of the patient group were significantly higher than those of the control group for the countermovement jump (15% increase), power during rebound jump (41%), work done in standing jump (12%) and countermovement jump (22%), and the difference between countermovement jump and standing jump (effect of adding eccentric component). Athletes with jumper's knee demonstrated better performance in jump tests than uninjured athletes, particularly in ballistic jumps involving eccentric force generation.

We report on a young male with moderate mental retardation, dysmorphic features, and language delay who is deleted for 7q31.1-7q31.31. His full karyotype is 46,XY,der(7)del(7)(q31.1q31.31)ins(10;7)(q24.3;q31.1q31.31)mat. This child had language impairment, including developmental verbal dyspraxia, but did not meet criteria for autism according to standardized ADOS testing. Our patient's deletion, which is the smallest reported deletion including FOXP2, adds to the body of evidence that supports the role of FOXP2 in speech and language impairment, but not in autism. A reported association between autism and deletions of WNT2, a gene also deleted in our patient, is likewise not supported by our case. Previously, fine mapping with microsatellites markers within in a large three-generation family, in which half the members had severe specific language impairment, aided the localization of the SPCH1 locus to 7q31 within markers D7S2459 (107.1 Mb) and D7S643 (120.5 Mb). Additionally, chromosome rearrangement of 7q31 and mutational analyses have supported the growing evidence that FOXP2, a gene within the SPCH1 region, is involved with speech and language development. It is unclear however whether the AUTS1 (autistic spectrum 1) locus, highly linked to 7q31, overlaps with the SPCH1 and FOXP2.

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [13C]bicarbonate, [14C]bicarbonate, and [13C]acetate were performed during different times of the day. [14C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. 13C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [13C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [13C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.

The approximately 3,450-million-year-old Strelley Pool Formation in Western Australia contains a reef-like assembly of laminated sedimentary accretion structures (stromatolites) that have macroscale characteristics suggestive of biological influence. However, direct microscale evidence of biology--namely, organic microbial remains or biosedimentary fabrics--has to date eluded discovery in the extensively-recrystallized rocks. Recently-identified outcrops with relatively good textural preservation record microscale evidence of primary sedimentary processes, including some that indicate probable microbial mat formation. Furthermore, we find relict fabrics and organic layers that covary with stromatolite morphology, linking morphologic diversity to changes in sedimentation, seafloor mineral precipitation, and inferred microbial mat development. Thus, the most direct and compelling signatures of life in the Strelley Pool Formation are those observed at the microscopic scale. By examining spatiotemporal changes in microscale characteristics it is possible not only to recognize the presence of probable microbial mats during stromatolite development, but also to infer aspects of the biological inputs to stromatolite morphogenesis. The persistence of an inferred biological signal through changing environmental circumstances and stromatolite types indicates that benthic microbial populations adapted to shifting environmental conditions in early oceans.

Methionine adenosyltransferase (MAT), a key enzyme in metabolism, catalyzes the synthesis of one of the most important and pivotal biological molecules, S-adenosyl-methionine. In every organism studied thus far, MAT exists in multiple forms; most are encoded by related, but distinct genes. Molecular and immunological studies revealed the presence of considerable conservation in the structure of MAT from different species; however, the various MAT isozymes differ in their physical and kinetic properties in ways that allow them to be regulated differently. Recent studies suggest that human MAT is composed of nonidentical subunits that can assume multiple states of aggregation, each with different kinetic characteristics. The tissue distribution of MAT isozymes and the ability of cells within the same tissue to switch between the different forms of MAT suggest that this mode of regulation is important for cellular function and differentiation. Therefore, understanding the regulation and structure-function relationship of this fascinating enzyme should help us clarify its role in biology and may provide us with tools to effectively manipulate its activity in clinical situations such as cancer, autoimmunity and organ transplantation.

REII is a Schizosaccharomyces pombe repression element located at the centromere-proximal end of the mat silent domain. Here we show that inversion of REII enhances silencing on its centromere-proximal side while suppressing silencing on its centromere-distal side. Transplacement of REII to a position 2.5 kb from its native locus extends the region of stringent repression to the new REII site. These results suggest that REII defines a mat silent domain boundary by acting preferentially toward its centromere-distal side. To investigate cooperation between REII and a K-region sequence that shares homology with the centromeric dg dh repeats (cen2 homology), we targeted combinations of these elements to an ectopic site and monitored expression of an adjacent reporter gene. Centromeric dh-like sequences conferred low-level silencing on the adjacent reporter gene, and REII, which did not display silencing activity on its own, enhanced cen2 homology-mediated silencing. Cooperation was also apparent at the mat locus, where deletion of REII impaired repression stability. We propose that REII and the cen2 homology play different yet complementary roles in silencing establishment and inheritance at the mat locus.

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.

High-intensity ultrasound has been used to treat Dunning R3327 prostatic adenocarcinoma implanted s.c. in Fischer Copenhagen rats. Focused ultrasound was generated with a 1-MHz transducer and energy was provided by a 7.5-kW power amplifier. Seventy-four rats were treated using two different sublines of Dunning tumor. Study 1 dealt with 49 rats with the Mat-Ly-Lu subline, treated with acoustic intensities ranging from 300 to 2750 W/cm2. Of the 49 rats in Study 1, 30 had complete tumor necrosis and 19 had no effect; of the 30 who had complete local tumor necrosis, 14 had local relapse, 9 had distance metastases to lung and nodes without local occurrence, and 7 remained free of tumor and were still alive 12 months after treatment. In Study 2, 25 rats with AT2 subline were treated with an intensity of 820 W/cm2. Similarly for Study 2, there was complete local tumor necrosis in 24 of 25 animals, with local regrowth in 7 of 24 and no recurrence of metastasis in the remaining 16 after a follow-up of 3 months. These results suggested that high-intensity focused ultrasound could be useful for the treatment of small localized cancerous tumors such as low-grade prostate carcinoma.

In the filamentous fungus Podospora anserina, mating type is specified by a single locus with two alternate alleles, termed mat- and mat+. A previous study has shown that the mat+ sequence consists of 3.7 kb and contains a single gene relevant to the sexual cycle. This gene, called FPR1, encodes a protein with a HMG DNA-binding domain and is required for fertilization and for the development of the fertilized fruiting body. The mat- sequence, which is 4.7 kb in length, displays a more complex structure. We present here the characterization of two genes, called SMR1 and SMR2, which are present in the mat- allele along with the FMR1 gene. FMR1, whose role in the sexual cycle has been already partially described, encodes a protein with an alpha 1-domain and was shown to control fertilization. We demonstrate that these three genes are required for the developmental events that occur in the female organ after fertilization. The additional role of FMR1 requires a region of unknown function that is distinct from the alpha 1-domain. SMR1 encodes a protein with a putative acidic/hydrophobic alpha-helix, which has been proposed to be a feature common to transcriptional activators. The protein sequence deduced from SMR2 contains an HMG motif suggesting that it is a transcription factor.

After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.

Saccharomyces mating-type switching results from replacement by gene conversion of the MAT locus with sequences copied from one of two unexpressed donor loci, HML or HMR. MATa cells recombine with HMLalpha approximately 90% of the time, whereas MATalpha cells choose HMRa 80%-90% of the time. HML preference in MATa is controlled by the cis-acting recombination enhancer (RE) that regulates recombination along the entire left arm of chromosome III. Comparison of RE sequences between S. cerevisiae, S. carlsbergensis, and S. bayanus defines four highly conserved regions (A, B, C, and D) within a 270-bp minimum RE. An adjacent E region enhances RE activity. Multimers of region A, D, or E are sufficient to promote selective use of HML. Regions A, D, and E each bind in vivo the transcription activator forkhead proteins Fkh1p and Fkh2p and their associated Ndd1p, although there are no adjacent open reading frames (ORFs). Deletion of FKH1 significantly reduces MATa's use of HML, as does mutation of the Fkh1/Fkh2-binding sites in a multimer of region A. We conclude that Fkh1p regulates MATa donor preference through direct interaction with RE.