Genome wide association in Spanish bread wheat landraces identifies six key genomic regions that constitute potential targets for improving grain yield related traits.

KEY MESSAGE: Association mapping conducted in 189 Spanish bread wheat landraces revealed six key genomic regions that constitute stable QTLs for yield and include 15 candidate genes. Genetically diverse landraces provide an ideal population to conduct association analysis. In this study, association mapping was conducted in a collection of 189 Spanish bread wheat landraces whose genomic diversity had been previously assessed. These genomic data were combined with characterization for yield-related traits, including grain size and shape, and phenological traits screened across five seasons. The association analysis revealed a total of 881 significant marker trait associations, involving 434 markers across the genome, that could be grouped in 366 QTLs based on linkage disequilibrium. After accounting for days to heading, we defined 33 high density QTL genomic regions associated to at least four traits. Considering the importance of detecting stable QTLs, 6 regions associated to several grain traits and thousand kernel weight in at least three environments were selected as the most promising ones to harbour targets for breeding. To dissect the genetic cause of the observed associations, we studied the function and in silico expression of the 413 genes located inside these six regions. This identified 15 candidate genes that provide a starting point for future analysis aimed at the identification and validation of wheat yield related genes.

Development and marker-trait relationships of functional markers for glutamine synthetase GS1 and GS2 homoeogenes in bread wheat.

GS1 and GS2 genes encode, respectively, the main cytosolic and the plastidic isoforms of glutamine synthetase (GS). In the present study, the wheat GS1 and GS2 homoeogenes located in the A, B and D genome chromosomes have been sequenced in a group of 15 bread wheat varieties including landraces, old commercial varieties and modern cultivars. Phenotypic characterization by multi-environment field trials detected significant effects of specific GS homoeogenes on three of the seven agronomic and grain quality traits analyzed. Based on the gene sequence polymorphisms found, biallelic molecular markers that could facilitate marker-assisted breeding were developed for genes GS1A, GS2A and GS2D. The remaining genes encoding main wheat GS were excluded because of being monomorphic (GS1D) or too polymorphic (GS1B and GS2B) in the sequencing panel varieties. A collection of 187 Spanish bread wheat landraces was genotyped for these gene-based molecular markers. Data analyses conducted with phenotypic records reported for this germplasm collection in López-Fernández et al. (Plants-Basel 10: 620, 2021) have revealed the beneficial influence of some individual alleles on thousand-kernel weight (TKW), kernels per spike (KS) and grain protein content. Furthermore, genetic interactions between GS1A, a cytosolic GS isoform coding gene, and GS2A or GS2D, plastidic GS enzyme coding genes, were found to affect TKW and KS. The finding that some alleles at one locus may mask the effect of positive alleles at hypostatic GS loci should be kept in mind if gene pyramiding strategies are attempted for the improvement of N-use efficiency-related traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01354-0.

Structural Characterization and Molecular Dynamics Study of the REPI Fusion Protein from Papaver somniferum L.

REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.

Exploring the End-Use Quality Potential of a Collection of Spanish Bread Wheat Landraces.

Modern plant-breeding practices have narrowed the genetic base of wheat, such that there is a need to introduce new germplasms with underexploited diversity into breeding programs. Wheat landraces are a very valuable resource when searching for genetic variation, which not only possess increased adaptability, but also quality-related traits. Several studies have shown a wide genetic diversity in Spanish wheat landraces compared to other germplasm collections; therefore, the main objective of this study is to analyze the variability in a collection of 189 landraces from the Spanish National Plant Genetic Resources Centre (Centro de Recursos Fitogenéticos, CRF-INIA, Alcalá de Henares), in relation to end-use quality traits. We characterized the whole collection for high-molecular-weight glutenin and puroindoline allelic composition, and for gluten strength. In addition, grain protein content, grains per spike, and thousand kernel weight were evaluated in samples from four-year field trials. The relationship between glutenin composition and quality was evaluated, and some alleles strongly associated with high quality were identified in the collection, some of them specific for Iberian landraces. The results also show the presence of novel variability within high-molecular-weight glutenin and puroindolines, which needs to be characterized further in order to assess its influence on wheat quality. In addition, a set of landraces showing outstanding values for gluten quality and a good agronomic performance was selected for testing in field trials in order to evaluate the suitability of their direct use in cropping systems.

An F2 Barley Population as a Tool for Teaching Mendelian Genetics.

In the context of a general genetics course, mathematical descriptions of Mendelian inheritance and population genetics are sometimes discouraging and students often have serious misconceptions. Innovative strategies in expositive classes can clearly encourage student's motivation and participation, but laboratories and practical classes are generally the students' favourite academic activities. The design of lab practices focused on learning abstract concepts such as genetic interaction, genetic linkage, genetic recombination, gene mapping, or molecular markers is a complex task that requires suitable segregant materials. The optimal population for pedagogical purposes is an F2 population, which is extremely useful not only in explaining different key concepts of genetics (as dominance, epistasis, and linkage) but also in introducing additional curricular tools, particularly concerning statistical analysis. Among various model organisms available, barley possesses several unique features for demonstrating genetic principles. Therefore, we generated a barley F2 population from the parental lines of the Oregon Wolfe Barley collection. The objective of this work is to present this F2 population as a model to teach Mendelian genetics in a medium-high-level genetics course. We provide an exhaustive phenotypic and genotypic description of this plant material that, together with a description of the specific methodologies and practical exercises, can be helpful for transferring our fruitful experience to anyone interested in implementing this educational resource in his/her teaching.

Genomic analysis of Spanish wheat landraces reveals their variability and potential for breeding.

BACKGROUND: One of the main goals of the plant breeding in the twenty-first century is the development of crop cultivars that can maintain current yields in unfavorable environments. Landraces that have been grown under varying local conditions include genetic diversity that will be essential to achieve this objective. The Center of Plant Genetic Resources of the Spanish Institute for Agriculture Research maintains a broad collection of wheat landraces. These accessions, which are locally adapted to diverse eco-climatic conditions, represent highly valuable materials for breeding. However, their efficient use requires an exhaustive genetic characterization. The overall aim of this study was to assess the diversity and population structure of a selected set of 380 Spanish landraces and 52 reference varieties of bread and durum wheat by high-throughput genotyping. RESULTS: The DArTseq GBS approach generated 10 K SNPs and 40 K high-quality DArT markers, which were located against the currently available bread and durum wheat reference genomes. The markers with known locations were distributed across all chromosomes with relatively well-balanced genome-wide coverage. The genetic analysis showed that the Spanish wheat landraces were clustered in different groups, thus representing genetic pools providing a range of allelic variation. The subspecies had a major impact on the population structure of the durum wheat landraces, with three distinct clusters that corresponded to subsp. durum, turgidum and dicoccon being identified. The population structure of bread wheat landraces was mainly biased by geographic origin. CONCLUSIONS: The results showed broader genetic diversity in the landraces compared to a reference set that included commercial varieties, and higher divergence between the landraces and the reference set in durum wheat than in bread wheat. The analyses revealed genomic regions whose patterns of variation were markedly different in the landraces and reference varieties, indicating loci that have been under selection during crop improvement, which could help to target breeding efforts. The results obtained from this work will provide a basis for future genome-wide association studies.

A Novel Emergency Department-based Community Notification Method for Clinical Research Without Consent.

OBJECTIVE: We used an emergency department (ED)-based method to provide targeted, individualized consultation; community notification; and public disclosure and collect data regarding willingness to participate in prospective resuscitation research requiring waiver of consent. METHODS: We conducted a prospective survey of convenience cohort in an urban ED. We targeted the community of ED patients with pulmonary disease for individualized notification and public disclosure using a 1) large poster, 2) scripted oral presentation describing an emergency intubation clinical trial, and 3) video demonstration. RESULTS: Approximately 10% of our annual ED census, 6,936 subjects, enrolled. Of that total, 29 were also subjects in a prospective coincident endotracheal resuscitation intubation study, which enrolled a total of 262 subjects. ED community notification was provided to 22 of the 29 (75.9%) subjects prior to the visit during which they were intubated (13 agreed to participate, six declined, and three undecided) and seven of the 29 subjects subsequent to enrollment in the intubation study (five agreed to participate and two undecided). Fourteen of the 29 patients who participated in both projects had undergone endotracheal intubation at least once prior to community notification: 10 agreed to participate in the study, two declined, and two were undecided. CONCLUSIONS: Emergency department-based community notification and public disclosure is a viable way to provide information to a target population and collect data about the success of the notification. Feedback data collection is critical to an ethical understanding of the success of community notification for the institutional review board and investigators. Collection of feedback data should be required as a subject protection for exception from informed consent in emergency settings.

Genetic Diversity and Association Mapping for Agromorphological and Grain Quality Traits of a Structured Collection of Durum Wheat Landraces Including subsp. durum, turgidum and diccocon.

Association mapping was performed for 18 agromorphological and grain quality traits in a set of 183 Spanish landraces, including subspecies durum, turgidum and dicoccon, genotyped with 749 DArT (Diversity Array Technology) markers. Large genetic and phenotypic variability was detected, being the level of diversity among the chromosomes and genomes heterogeneous, and sometimes complementary, among subspecies. Overall, 356 were monomorphic in at least one subspecies, mainly in dicoccon, and some of them coincidental between subspecies, especially between turgidum and dicoccon. Several of those fixed markers were associated to plant responses to environmental stresses or linked to genes subjected to selection during tetraploid wheat domestication process. A total of 85 stable MTAs (marker-trait associations) have been identified for the agromorphological and quality parameters, some of them common among subspecies and others subspecies-specific. For all the traits, we have found MTAs explaining more than 10% of the phenotypic variation in any of the three subspecies. The number of MTAs on the B genome exceeded that on the A genome in subsp. durum, equalled in turgidum and was below in dicoccon. The validation of several adaptive and quality trait MTAs by combining the association mapping with an analysis of the signature of selection, identifying the putative gene function of the marker, or by coincidences with previous reports, showed that our approach was successful for the detection of MTAs and the high potential of the collection to identify marker-trait associations. Novel MTAs not previously reported, some of them subspecies specific, have been described and provide new information about the genetic control of complex traits.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range.

UNLABELLED: • PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses (B. distachyon, B. stacei) and their derived allotetraploid (B. hybridum), native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identified potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range.• METHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene flow among the hybridizing species of the complex.• KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth.• CONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversification was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon.

Brachypodium distachyon (L.) P. Beauv. (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20, and 30 chromosomes) has long been regarded as a case of autopolyploid series with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as Brachypodium stacei (2n = 2x = 20) and Brachypodium hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i.e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers 83 B. distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4, and 15 had 10, 20, and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with nonoverlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.

Can intraocular pressure measurements be used to screen for elevated intracranial pressure in emergency department patients?

BACKGROUND: Handheld measurement of intraocular pressure (IOP) has been previously shown to accurately predict elevated intracranial pressure (ICP) in neurosurgical patients. Handheld tonometry may have clinical utility in the prediction of elevated ICP among a cohort of emergency department (ED) patients receiving lumbar puncture (LP). OBJECTIVE: To ascertain the sensitivity and specificity of IOP for the prediction of elevated ICP in ED patients undergoing LP. METHODS: In this prospective observational pilot study, all ED patients over the age of 18 years and undergoing LP in the ED for any reason were eligible to participate. Study participants had IOP measured with the Tono-Pen XL (Reichert, Inc., Depew, NY) while in the supine position before LP. OP was measured in the lateral recumbent position. Elevated IOP was defined as≥20mm Hg; elevated ICP was defined as≥20mm H(2)O. RESULTS: There were 82 patients screened and 46 patients enrolled at the time of interim analysis. Of the 46 patients, 32 had a successful LP in the lateral recumbent position. There were 18/32 patients with a successful LP in the lateral recumbent position who had elevated opening pressure; 9/32 patients with a successful LP had an elevated IOP. Furthermore, 4/9 patients with elevated IOP also had an elevated opening pressure. There was only one patient who had elevated IOP, elevated ICP, and diagnostic cerebrospinal fluid (sensitivity 24%, 95% confidence interval [CI] 9-48%; specificity 63%, 95% CI 32-88%; positive predictive value 28%, 95% CI 14-47%; negative predictive value 72%, 95% CI 53-96%). CONCLUSIONS: Handheld tonometry has poor sensitivity and specificity for the prediction of increased ICP, and should not be used as a screening tool in the ED.

Two days of dexamethasone versus 5 days of prednisone in the treatment of acute asthma: a randomized controlled trial.

STUDY OBJECTIVE: Dexamethasone has a longer half-life than prednisone and is well tolerated orally. We compare the time needed to return to normal activity and the frequency of relapse after acute exacerbation in adults receiving either 5 days of prednisone or 2 days of dexamethasone. METHODS: We randomized adult emergency department patients (aged 18 to 45 years) with acute exacerbations of asthma (peak expiratory flow rate less than 80% of ideal) to receive either 50 mg of daily oral prednisone for 5 days or 16 mg of daily oral dexamethasone for 2 days. Outcomes were assessed by telephone follow-up. RESULTS: Ninety-six prednisone and 104 dexamethasone subjects completed the study regimen and follow-up. More patients in the dexamethasone group reported a return to normal activities within 3 days compared with the prednisone group (90% versus 80%; difference 10%; 95% confidence interval 0% to 20%; P=.049). Relapse was similar between groups (13% versus 11%; difference 2%; 95% confidence interval -7% to 11%, P=.67). CONCLUSION: In acute exacerbations of asthma in adults, 2 days of oral dexamethasone is at least as effective as 5 days of oral prednisone in returning patients to their normal level of activity and preventing relapse.

Routine packing of simple cutaneous abscesses is painful and probably unnecessary.

OBJECTIVES: The objective was to determine whether the routine packing of simple cutaneous abscesses after incision and drainage (I&D) confers any benefit over I&D alone. METHODS: In a prospective, randomized, single-blinded trial, subjects with simple cutaneous abscesses (less than 5 cm largest diameter) underwent incision, drainage, irrigation, and standard abscess preparation in the usual manner. Subjects were then randomized to either packing or no-packing. Visual analog scales (VAS; 100 mm) of pain were recorded in the emergency department (ED). All patients received trimethoprim-sulfamethoxazole (TMP-SMX), ibuprofen, and narcotic prescriptions, recorded twice daily VAS pain scores, and returned in 48 hours at which time dressings and packing, if present, were removed and a physician blinded to the randomization and not part of the initial visit repeated measurements and determined the need for further intervention. RESULTS: Forty-eight subjects were included in the final analysis. There were no significant differences in age, sex, abscess location, or initial pain scores between the two groups. There was no significant difference in need for a second intervention at the 48-hour follow-up between the packed (4 of 23 subjects) and nonpacked (5 of 25 subjects) groups (p = 0.72; relative risk = 1.3, 95% confidence interval [CI] = 0.4 to 4.2). Patients in the group that received packing reported higher pain scores immediately postprocedure (mean difference = 23.8 mm; p = 0.014, 95% CI = 5 to 42 mm) and at 48 hours postprocedure (mean difference = 16.4 mm; p = 0.03, 95% CI = 1.6 to 31.2 mm), as well as greater use of ibuprofen (mean difference = 0.32; p = 0.12, 95% CI = -1.4 to 2.0) and oxycodone/acetaminophen (mean difference = 2.19; p = 0.03, 95% CI = 0.2 to 4.1). CONCLUSION: In this pilot study, not packing simple cutaneous abscesses did not result in any increased morbidity, and patients reported less pain and used fewer pain medications than packed patients.

Eficacia del colgajo uvulopalatal extendio en el manejo del ronquido simple / Efficacy of extended uvulopalatal flap for the treatment of simple snoring

El ronquido hace parte del espectro de los desórdenes respiratorios del sueño, es una entidad común,que afecta 30 porciento de los adultos; a pesar del múltiple desarrollo de terapias para su manejo, no se ha encontrado el tratamiento idealObjetivo: Determinar si hay mejoría significativa en el ronquido luego de realizar el colgajo uvulopalatal extendido.Materiales y métodos: Estudio prospectivo, donde se evaluaron 24 pacientes con diagnóstico de roncopatía, a los cuales se les realizó el colgajo uvulopalatal extendido y 6 meses posquirúrgicos, se evaluó la escala visual análoga (EVA) para ronquido, la escala de epworth y el índice de masa corporal (IMC).Resultados: Se encontró mejoría significativa del ronquido en 16/24 pacientes (67 porciento), si excluimos los pacientes con hipertrofia de base de lengua, la mejoría es en 14/19 pacientes (74 porciento), asociado a esto se encontró una mejoría en la escala de epworth en 20/24 pacientes (83 porciento). Estas diferencias fueron estadísticamente significativas (p< 0,05)Conclusiones: El colgajo uvulopalatal extendido es una buena alternativa para el manejo del ronquido.

Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection.

Genomic-type transgenes are usually expressed in appropriate spatial- and temporal-specific manners. The largest genomic transgenes can be prepared using yeast artificial chromosomes (YACs). Normally, YAC transgenic mice are produced by standard pro-nuclear microinjection, although other methods, involving the use of embryonic stem (ES) cells, have been also devised. To overcome the difficulty and time extension of ES cell-type approaches and to improve the rather usual low efficiency of YAC DNA transgenesis by pronuclear microinjection, that is mostly dependent on the YAC DNA quality of samples, we have devised an updated intracytoplasmic sperm injection (ICSI) method for the stable incorporation of YACs into the germ line of mice. DNA transgenesis efficiencies achieved are often 10 times greater than those usually obtained by standard microinjection, thus enabling the identification of either more transgenic founder animals and the use of reduced numbers of individuals in animal experimentation.

Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region of Ontario.

To determine the prevalence of Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region, Ontario, 5 of 26 clinics invited by mail survey reported their parasitological findings over 24 months. Stool samples collected by 1 clinic over 68 days were investigated for parasites by using several techniques (fecal concentration, acid-fast staining, and a Cryptosporidium immunoassay). The 5 clinics that provided data indicated Toxocara spp. as the most frequent finding. Parasitological study of 111 stool samples showed a high overall positivity rate in samples from both dogs (40%) and cats (36.6%). Cryptosporidium spp. antigen was detected in 7.4% and 7.3%, Toxocara spp. in 14.2% and 12.2%, and Giardia spp. 7.1% and 2.4% of dog and cat samples, respectively. The high prevalence of zoonotic parasites in the Niagara region is important, and increased awareness of their potential threat to human health is necessary. Additionally, further research into the zoonotic capacity of Cryptosporidium spp. and Giardia spp. is necessary.

Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection.

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism.

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.

Identification and functional validation of a 5' upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene.

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non-coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5' upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at -15 kb. We detected several stretches of homology within the first 30 kb 5' tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5' upstream regulatory sequence found between -8 and -10 kb of the human tyrosinase locus (termed h5'URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at -9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue-specific enhancer in the h5'URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5'URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.

Tyrosinase gene expression is not detected in mouse brain outside the retinal pigment epithelium cells.

Tyrosinase is the rate-limiting enzyme for melanin synthesis. Its gene is expressed in two cell types: melanocytes, derived from migrating neural crest cells, and, in the CNS, retinal pigment epithelium cells, derived from the optic cup. Its absence from the eye results in profound pathway selection errors of optic fibres at the chiasm and, hence, it has been implicated as a developmental regulator of CNS pathway selection. Recently, it has been proposed that tyrosinase can also be expressed in the developing and adult brain, although the methods used were indirect. Its presence in the brain could be very significant in terms of a potentially wider role in pathway finding. Here, we have evaluated the presence of tyrosinase expression in mouse developing, perinatal and adult brain by in situ hybridization in whole-mount embryos and histological sections and by real-time reverse transcription-polymerase chain reaction. We find no evidence for tyrosinase gene expression in the CNS outside the retinal pigment epithelium cells.