Identification of genomic regions associated with total and progressive sperm motility in Italian Holstein bulls.

Sperm motility is directly related to the ability of sperm to move through the female reproductive tract to reach the ovum. Sperm motility is a complex trait that is influenced by environmental and genetic factors and is associated with male fertility, oocyte penetration rate, and reproductive success of cattle. In this study we carried out a GWAS in Italian Holstein bulls to identify candidate regions and genes associated with variations in progressive and total motility (PM and TM, respectively). After quality control, the final data set consisted of 5,960 records from 949 bulls having semen collected in 10 artificial insemination stations and genotyped at 412,737 SNPs (call rate >95%; minor allele frequency >5%). (Co)variance components were estimated using single trait mixed models, and associations between SNPs and phenotypes were assessed using a genomic BLUP approach. Ten windows that explained the greatest percentage of genetic variance were located on Bos taurus autosomes 1, 2, 4, 6, 7, 23, and 26 for TM and Bos taurus autosomes 1, 2, 4, 6, 8, 16, 23, and 26 for PM. A total of 150 genes for TM and 72 genes for PM were identified within these genomic regions. Gene Ontology enrichment analyses identified significant Gene Ontology terms involved with energy homeostasis, membrane functions, sperm-egg interactions, protection against oxidative stress, olfactory receptors, and immune system. There was significant enrichment of quantitative trait loci for fertility, calving ease, immune response, feed intake, and carcass weight within the candidate windows. These results contribute to understanding the architecture of the genetic control of sperm motility and may aid in the development of strategies to identify subfertile bulls and improve reproductive success.

Asian water buffalo: domestication, history and genetics.

The domestic Asian water buffalo (Bubalus bubalis) is found on all five continents, with a global population of some 202 million. The livelihoods of more people depend on this species than on any other domestic animal. The two distinct types (river and swamp) descended from different wild Asian water buffalo (Bubalus arnee) populations that diverged some 900 kyr BP and then evolved in separate geographical regions. After domestication in the western region of the Indian subcontinent (ca. 6300 years BP), the river buffalo spread west as far as Egypt, the Balkans and Italy. Conversely, after domestication in the China/Indochina border region ca. 3000-7000 years BP, swamp buffaloes dispersed through south-east Asia and China as far as the Yangtze River valley. Molecular and morphological evidence indicates that swamp buffalo populations have strong geographic genetic differentiation and a lack of gene flow, but strong phenotypic uniformity. In contrast, river buffalo populations show a weaker phylogeographic structure, but higher phenotypic diversity (i.e. many breeds). The recent availability of a high-quality reference genome and of a medium-density marker panel for genotyping has triggered a number of genome-wide investigations on diversity, evolutionary history, production traits and functional elements. The growing molecular knowledge combined with breeding programmes should pave the way to improvements in production, environmental adaptation and disease resistance in water buffalo populations worldwide.

Inbreeding and purging at the genomic Level: the Chillingham cattle reveal extensive, non-random SNP heterozygosity.

Local breeds of livestock are of conservation significance as components of global biodiversity and as reservoirs of genetic variation relevant to the future sustainability of agriculture. One such rare historic breed, the Chillingham cattle of northern England, has a 350-year history of isolation and inbreeding yet shows no diminution of viability or fertility. The Chillingham cattle have not been subjected to selective breeding. It has been suggested previously that the herd has minimal genetic variation. In this study, high-density SNP genotyping with the 777K SNP chip showed that 9.1% of loci on the chip are polymorphic in the herd, compared with 62-90% seen in commercial cattle breeds. Instead of being homogeneously distributed along the genome, these loci are clustered at specific chromosomal locations. A high proportion of the Chillingham individuals examined were heterozygous at many of these polymorphic loci, suggesting that some loci are under balancing selection. Some of these frequently heterozygous loci have been implicated as sites of recessive lethal mutations in cattle. Linkage disequilibrium equal or close to 100% was found to span up to 1350 kb, and LD was above r(2) = 0.25 up to more than 5000 kb. This strong LD is consistent with the lack of polymorphic loci in the herd. The heterozygous regions in the Chillingham cattle may be the locations of genes relevant to fitness or survival, which may help elucidate the biology of local adaptation in traditional breeds and facilitate selection for such traits in commercial cattle.

Multilocus genotypic data reveal high genetic diversity and low population genetic structure of Iranian indigenous sheep.

Iranian livestock diversity is still largely unexplored, in spite of the interest in the populations historically reared in this country located near the Fertile Crescent, a major livestock domestication centre. In this investigation, the genetic diversity and differentiation of 10 Iranian indigenous fat-tailed sheep breeds were investigated using 18 microsatellite markers. Iranian breeds were found to host a high level of diversity. This conclusion is substantiated by the large number of alleles observed across loci (average 13.83, range 7-22) and by the high within-breed expected heterozygosity (average 0.75, range 0.72-0.76). Iranian sheep have a low level of genetic differentiation, as indicated by the analysis of molecular variance, which allocated a very small proportion (1.67%) of total variation to the between-population component, and by the small fixation index (FST  = 0.02). Both Bayesian clustering and principal coordinates analysis revealed the absence of a detectable genetic structure. Also, no isolation by distance was observed through comparison of genetic and geographical distances. In spite of high within-breed variation, signatures of inbreeding were detected by the FIS indices, which were positive in all and statistically significant in three breeds. Possible factors explaining the patterns observed, such as considerable gene flow and inbreeding probably due to anthropogenic activities in the light of population management and conservation programmes, are discussed.

A method for single nucleotide polymorphism selection for parentage assessment in goats.

Accurate pedigrees are essential to optimize genetic improvement and conservation of animal genetic resources. In goats, the use of mating groups and kidding management procedures hamper the identification of parentage. Small panels of single nucleotide polymorphisms (SNP) have been proposed in other species to substitute microsatellites for parentage assessment. Using data from the current GoatSNP50 chip, we developed a new 3-step procedure to identify a low-density SNP panel for highly accurate parentage assessment. Methodologies for SNP selection used in other species are less suitable in the goat because of uncertainties in the genome assembly. The procedure developed in this study is based on parent-offspring identification and on estimation of Mendelian errors, followed by canonical discriminant analysis identification and stepwise regression reduction. Starting from a reference sample of 109 Alpine goats with known pedigree relationships, we first identified a panel of 200 SNP that was further reduced to 2 final panels of 130 and 114 SNP with random coincidental match inclusion of 1.51×10(-57) and 2.94×10(-34), respectively. In our reference data set, all panels correctly identified all parent-offspring combinations, revealing a 40% pedigree error rate in the information provided by breeders. All reference trios were confirmed by official tests based on microsatellites. Panels were also tested on Saanen and Teramana breeds. Although the testing on a larger set of breeds in the reference population is still needed to validate these results, our findings suggest that our procedure could identify SNP panels for accurate parentage assessment in goats or in other species with unreliable marker positioning.

Detecting population structure and recent demographic history in endangered livestock breeds: the case of the Italian autochthonous donkeys.

Since its domestication, about 5000 years ago, the donkey (Equus asinus) has been extensively used as a work or draft animal in agricultural activities and for the transportation of people and goods. In the last century, technology improvement and growing mechanization strongly affected agriculture and the management and use of this livestock species in the industrialized countries. Nowadays, the use of donkeys for work or transport has almost disappeared, together with the need for mules or hinny breeding. During the last five decades, Italian autochthonous donkey populations suffered from a severe reduction in population size, which led to the extinction of several breeds. At present, eight breeds remain, all classified by FAO as critically endangered or endangered: Asinara, Pantesco, Grigio Siciliano, Romagnolo, Amiatino, Sardo Grigio, Martina Franca, and Ragusano. To evaluate the extant genetic variability of Italian donkeys, we typed 16 microsatellite loci in 258 individuals from these breeds. The results highlighted moderate levels of inbreeding ( F (IS) = 0.127) and a significant partition of genetic variation into breeds, as suggested by fixation index ( F (ST) = 0.109) and analysis of molecular variance (10.86% of total variation assigned to the between-breeds level) analyses. This was confirmed by a Bayesian clustering procedure that also highlighted a further partitioning at lower hierarchical levels corresponding to the farms of origin. This evidence suggests that an effective management strategy for Italian donkey populations should focus on breeds as conservation units. However, this requires a synergic management strategy at the farm level to maintain diversity and avoid inbreeding.

Social and behavioral determinants as risk of sexually transmitted diseases. Report by a sample from the Sexually Transmitted Disease Unit in Florence, Italy.

AIM: Sexually transmitted infections (STIs) are increasing worldwide, mostly due to changing sexual behavior s (larger numbers of sexual partners, concurrent relationships, increasing proportion of adolescents engaging in sexual intercourse at young age, and inconsistent condom use with new partners). In Italy, few data are available about STI spread, since most infections are not subjected to mandatory notification. METHODS: In this article, the occurrence of STIs in a random sample attending a STI Unit in Florence, Italy, is reported. Results were obtained through the administration of an anonymous questionnaire that patients could complete spontaneously in the waiting room while waiting for the visit. Self-reported questions allowed to collect information about socio-demographic and clinical data, sexual behavior and perception of risk. RESULTS: Overall, 469 patients (321 males, 148 females) participated in the study. Age ranged from 16 to 70 years. Male patients who referred to engage sexual intercourse with men (MSM) were 133; females who had sex with women (FSF) were 5, while 24 patients declared to have sex with both males and females (bisexual); 59.7% (N.=280) of participants reported they had a stable relationship, but 20% of these reported they had had sex with more than five partners during the last 12 months. The use of condoms is declared to be very infrequent, especially in the two extreme age ranges. Fifty percent of patients had been diagnosed an STI in their life, particularly syphilis (39.3%), genital warts (64.6%) and chlamydial infections (42.9%). Among those subjects who had contracted an STI (including non-curable viral infections, i.e., HIV and herpes genitalis) 32.4% referred they never used condoms. CONCLUSION: The authors discuss their results compared to the existing literature, and focus on identification of risk factors associated with self-reported STIs. Although conducted on a small population, this study provides a basis for targeting prevention and control strategies on our high-risk patients.

Integrating geo-referenced multiscale and multidisciplinary data for the management of biodiversity in livestock genetic resources.

In livestock genetic resource conservation, decision making about conservation priorities is based on the simultaneous analysis of several different criteria that may contribute to long-term sustainable breeding conditions, such as genetic and demographic characteristics, environmental conditions, and role of the breed in the local or regional economy. Here we address methods to integrate different data sets and highlight problems related to interdisciplinary comparisons. Data integration is based on the use of geographic coordinates and Geographic Information Systems (GIS). In addition to technical problems related to projection systems, GIS have to face the challenging issue of the non homogeneous scale of their data sets. We give examples of the successful use of GIS for data integration and examine the risk of obtaining biased results when integrating datasets that have been captured at different scales.

Objectives, criteria and methods for using molecular genetic data in priority setting for conservation of animal genetic resources.

The genetic diversity of the world's livestock populations is decreasing, both within and across breeds. A wide variety of factors has contributed to the loss, replacement or genetic dilution of many local breeds. Genetic variability within the more common commercial breeds has been greatly decreased by selectively intense breeding programmes. Conservation of livestock genetic variability is thus important, especially when considering possible future changes in production environments. The world has more than 7500 livestock breeds and conservation of all of them is not feasible. Therefore, prioritization is needed. The objective of this article is to review the state of the art in approaches for prioritization of breeds for conservation, particularly those approaches that consider molecular genetic information, and to identify any shortcomings that may restrict their application. The Weitzman method was among the first and most well-known approaches for utilization of molecular genetic information in conservation prioritization. This approach balances diversity and extinction probability to yield an objective measure of conservation potential. However, this approach was designed for decision making across species and measures diversity as distinctiveness. For livestock, prioritization will most commonly be performed among breeds within species, so alternatives that measure diversity as co-ancestry (i.e. also within-breed variability) have been proposed. Although these methods are technically sound, their application has generally been limited to research studies; most existing conservation programmes have effectively primarily based decisions on extinction risk. The development of user-friendly software incorporating these approaches may increase their rate of utilization.

Assessing SNP markers for assigning individuals to cattle populations.

The effectiveness of single nucleotide polymorphisms (SNPs) for the assignment of cattle to their source breeds was investigated by analysing a panel of 90 SNPs assayed on 24 European breeds. Breed assignment was performed by comparing the Bayesian and frequentist methods implemented in the STRUCTURE 2.2 and GENECLASS 2 software programs. The use of SNPs for the reallocation of known individuals to their breeds of origin and the assignment of unknown individuals was tested. In the reallocation tests, the methods implemented in STRUCTURE 2.2 performed better than those in GENECLASS 2, with 96% vs. 85% correct assignments respectively. In contrast, the methods implemented in GENECLASS 2 showed a greater correct assignment rate in allocating animals treated as unknowns to a reference dataset (62% vs. 51% and 80% vs. 65% in field tests 1 and 2 respectively). These results demonstrate that SNPs are suitable for the assignment of individuals to reference breeds. The results also indicate that STRUCTURE 2.2 and GENECLASS 2 can be complementary tools to assess breed integrity and assignment. Our findings also stress the importance of a high-quality reference dataset in allocation studies.

High performance computation of landscape genomic models including local indicators of spatial association.

With the increasing availability of both molecular and topo-climatic data, the main challenges facing landscape genomics - that is the combination of landscape ecology with population genomics - include processing large numbers of models and distinguishing between selection and demographic processes (e.g. population structure). Several methods address the latter, either by estimating a null model of population history or by simultaneously inferring environmental and demographic effects. Here we present samßada, an approach designed to study signatures of local adaptation, with special emphasis on high performance computing of large-scale genetic and environmental data sets. samßada identifies candidate loci using genotype-environment associations while also incorporating multivariate analyses to assess the effect of many environmental predictor variables. This enables the inclusion of explanatory variables representing population structure into the models to lower the occurrences of spurious genotype-environment associations. In addition, samßada calculates local indicators of spatial association for candidate loci to provide information on whether similar genotypes tend to cluster in space, which constitutes a useful indication of the possible kinship between individuals. To test the usefulness of this approach, we carried out a simulation study and analysed a data set from Ugandan cattle to detect signatures of local adaptation with samßada, bayenv, lfmm and an FST outlier method (FDIST approach in arlequin) and compare their results. samßada - an open source software for Windows, Linux and Mac OS X available at http://lasig.epfl.ch/sambada - outperforms other approaches and better suits whole-genome sequence data processing.

Sonic Hedgehog pathway is upregulated in adamantinomatous craniopharyngiomas.

OBJECTIVES: Pituitary stem cells play a role in the oncogenesis of human adamantinomatous craniopharyngiomas (aCPs). We hypothesized that crosstalk between the Wnt/ß-catenin and Sonic Hedgehog (SHH) pathways, both of which are important in normal pituitary development, would contribute to the pathogenesis of aCPs. DESIGN: To explore the mRNA and protein expression of components of the SHH signaling pathway in aCPs and their relationship with the identification of CTNNB1/ß-catenin mutations and patients outcomes. PATIENTS AND METHODS: In 18 aCP samples, CTNNB1 was sequenced, and the mRNA expression levels of SHH pathway members (SHH, PTCH1, SMO, GLI1, GLI2, GLI3, and SUFU) and SMO, GLI1, GLI3, SUFU, ß-catenin, and Ki67 proteins were evaluated by quantitative real-time PCR and immunohistochemistry respectively. Anterior normal pituitaries were used as controls. Associations between molecular findings and clinical data were analyzed. RESULTS: The aCPs presented higher mRNA expression of SHH (+400-fold change (FC); P<0.01), GLI1 (+102-FC; P<0.001), and GLI3 (+5.1-FC; P<0.01) than normal anterior pituitaries. Longer disease-free survival was associated with low SMO and SUFU mRNA expression (P<0.01 and P=0.02 respectively). CTNNB1/ß-catenin mutations were found in 47% of the samples. aCPs with identified mutations presented with higher mRNA expression of SMO and GLI1 (+4.3-FC; P=0.02 and +10.2-FC; P=0.03 respectively). SMO, GLI1, GLI3, and SUFU staining was found in 85, 67, 93, and 64% of the samples respectively. Strong GLI1 and GLI3 staining was detected in palisade cells, which also labeled Ki67, a marker of cell proliferation. CONCLUSIONS: The upregulation of SHH signaling occurs in aCPs. Thus, activation of Wnt/ß-catenin and SHH pathways, both of which are important in pituitary embryogenesis, appears to contribute to the pathogenesis of aCP.

Membrane-bound choline acetyltransferase in Torpedo electric organ: a marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)

Amphiphilic and hydrophilic forms of choline-O-acetyltransferase in cholinergic nerve endings of the Torpedo.

In the purely cholinergic nerve endings isolated (i.e. synaptosomes) from the electric organ of the fish Torpedo, the enzyme choline acetyltransferase was found to exist not solely in its well-known soluble form but also in a form which is non-ionically bound to the plasma membrane; this activity could not be solubilized in solutions of high ionic strength (0.5 M NaCl). The non-ionic detergent Triton X-114 was used to solubilize synaptosomes isolated from either the electric organ of Torpedo or rat brain. This detergent allows to separate hydrophilic from amphiphilic proteins of cells or subcellular fractions. Twelve per cent of the synaptosomal choline acetyltransferase partitioned as amphiphilic and 80-97% as hydrophilic activity. The percentage of amphiphilic activity present in synaptosomes was significantly higher than that of the form of activity (4.4%) extracted from samples containing only the soluble form of choline acetyltransferase but was significantly lower than the percentage of amphiphilic enzyme present in preparations of synaptosomal plasma membrane (20-22%) which were enriched in the non-ionically membrane-bound form of choline acetyltransferase. These results indicate that the soluble and the non-ionically membrane-bound enzymes differ in their capacity to interact with non-ionic detergents. The preparations of synaptosomal plasma membranes contained significantly higher proportions of detergent-insoluble choline acetyltransferase activity than did the whole synaptosomes; the difference was more striking for the Torpedo than for the rat enzyme. This detergent-insoluble activity was not due to aggregates of the enzyme. Some properties of the hydrophilic and amphiphilic choline acetyltransferase of Torpedo were analyzed. The two forms of the enzyme did not exhibit different affinities for their substrates; they were found to differ with respect to their sensitivity to inhibition by increasing concentrations of the two products of the reaction, acetylcholine and coenzyme A and heat inactivation at 45 degrees C. Most probably the hydrophilic and amphiphilic activities correspond to what was referred to as soluble and non-ionically membrane-bound choline acetyltransferase, respectively. The amphiphilic form may be an integral enzyme of the plasma membrane of cholinergic nerve endings or may be tightly bound to a specific protein in this membrane which may act as a "receptor" for choline acetyltransferase.

Modulation of choline acetyltransferase synthesis by okadaic acid, a phosphatase inhibitor, and KN-62, a CaM kinase inhibitor, in NS-20Y neuroblastoma.

Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

Specific antibodies to bovine choline acetyltransferase raised in mice immunised with small amounts of partially purified enzyme.

Choline acetyltransferase was purified approximately 18,000-fold from 300 g of bovine caudate nuclei to a specific activity of 21 ?mol min mg protein. The overall procedure used was: extraction of the enzyme by high salt concentration, chromatography on carboxy-methyl-Sephadex, precipitation by ammonium sulphate, affinity chromatography on Blue-Sepharose and, finally absorption on hydroxylapatite. When the enzyme absorbed on hydroxylapatite was injected into mice, it provoked reproducibly a transient production of 'inhibitory' antibodies, followed by higher antibody titres mainly of 'non-inhibitory' type. These responses were elicited by injecting less than a total of 20 ?g of immunogen. The highest antibody titre was obtained less than 2 months following the initial immunisation. Species cross reactivity was investigated. This procedure should prove to be of value in the production of monoclonal antibodies to choline acetyltransferase.

Direct inhibition of choline acetyltransferase activity by a monoclonal antibody raised against the plasma membrane of cholinergic nerve terminals.

A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its Km during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.

Membrane-bound choline acetyltransferase of the torpedo has characteristics of an integral membrane protein and can be solubilized by proteolysis.

Due to Triton X-114 fractionation of synaptosomes isolated from the electric organ of the fish Torpedo, the existence of a hydrophilic and an amphiphilic form of the enzyme choline-O-acetyltransferase (ChAT) was revealed. Amphiphilic ChAT which represents about 10% of total enzyme activity in synaptosomes, reached 40% of ChAT activity measured in preparations of synaptosomal plasma membranes (SPM) which were washed with solutions of increasing ionic strength. ChAT activity bound to washed SPM could be partially solubilized using proteinase K but not phospholipase C. No ChAT solubilization occurred by treating intact synaptosomes with proteinase K. Water/Triton X-114 partition coefficients of hydrophilic and amphiphilic ChAT were found to be 6.5 and 0.17, respectively. Sedimentation coefficients determined by centrifugation in linear density gradients of sucrose containing Triton X-100, were 4.2S and 4.4S for amphiphilic and hydrophilic ChAT, respectively. On the other hand, removal of Triton X-114 from the detergent phase containing amphiphilic ChAT activity led to enzyme aggregation. Finally, amphiphilic ChAT was slightly more acidic (pH 6.6) than was hydrophilic enzyme (6.8-7.0). We conclude that in Torpedo synaptosomes two forms of ChAT activity, a soluble and a membrane-bound form, are indeed present which differ in their hydrophobicity. The soluble form is hydrophilic. The membrane-bound form is amphiphilic and it aggregates upon removal of detergent. These are two characteristics of integral membrane proteins. Membrane-bound ChAT is most probably intracellularly oriented and not bound to membrane through a 'receptor' protein.

The proportion of amphiphilic choline acetyltransferase in Drosophila melanogaster is higher than in rat or Torpedo and is developmentally regulated.

We show that in the central nervous system of the fly, Drosophila melanogaster, choline acetyltransferase (ChAT) activity exists under two molecular forms, a soluble, hydrophilic form and a membrane-bound, amphiphilic form. This is based on the following demonstrations of differential solubilization and interaction with non-denaturing detergents: sequential extraction of Drosophila heads produced low-salt-soluble (83-87%) and detergent-soluble (6-7%) ChAT activity. Sedimentation in sucrose gradients of detergent-soluble ChAT was found to be influenced by the type of detergent present in the gradient (Triton X-100 and Brij 96). This was not the case for low-salt-soluble ChAT. To further confirm these findings, we subjected Drosophila heads to Triton X-114 fractionation. This method, which yielded 12% of amphiphilic ChAT activity, separates hydrophilic from amphiphilic proteins. Compared to central nervous tissue of rat and Torpedo electric lobes, Drosophila head contained the highest proportion of amphiphilic ChAT activity. Synaptosomes isolated from Torpedo electric organ exhibited higher levels of amphiphilic ChAT than did electric lobes. Of the three animal species analyzed here, the Torpedo amphiphilic enzyme was the most hydrophobic and the rat enzyme the least hydrophobic. The proportion of amphiphilic ChAT was analyzed during Drosophila development. The percentage of this activity increased about 7 times from embryo to larva and then remained constant until the adult fly age.