Identification of putative adhesins and carbohydrate ligands of Lactobacillus paracasei using a combinatorial in silico and glycomics microarray profiling approach.

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 â†’ 3)-linked sialic acid and α-(1 â†’ 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT.

Engineering of CHO cells for the production of vertebrate recombinant sialyltransferases.

BACKGROUND: Sialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell-cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties. METHODS: The soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA). RESULTS: A range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format. CONCLUSION: Stable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme.

Glycosylation-related gene expression in HT29-MTX-E12 cells upon infection by Helicobacter pylori.

AIM: To identify glycosylation-related genes in the HT29 derivative cell line, HT29-MTX-E12, showing differential expression on infection with Helicobacter pylori (H. pylori). METHODS: Polarised HT29-MTX-E12 cells were infected for 24 h with H. pylori strain 26695. After infection RNA was isolated from both infected and non-infected host cells. Sufficient infections were carried out to provide triplicate samples for microarray analysis and for qRT-PCR analysis. RNA was isolated and hybridised to Affymetrix arrays. Analysis of microarray data identified genes significantly differentially expressed upon infection. Genes were grouped into gene ontology functional categories. Selected genes associated with host glycan structure (glycosyltransferases, hydrolases, lectins, mucins) were validated by real-time qRT-PCR analysis. RESULTS: Infection of host cells was confirmed by the isolation of live bacteria after 24 h incubation and by PCR amplification of bacteria-specific genes from the host cell RNA. H. pylori do not survive incubation under the adopted culture conditions unless they associate with the adherent mucus layer of the host cell. Microarray analysis identified a total of 276 genes that were significantly differentially expressed (P < 0.05) upon H. pylori infection and where the fold change in expression was greater than 2. Six of these genes are involved in glycosylation-related processes. Real-time qRT-PCR demonstrated significant downregulation (1.8-fold, P < 0.05) of the mucin MUC20. REG4 was heavily expressed and significantly downregulated (3.1-fold, P < 0.05) upon infection. Gene ontology analysis was consistent with previous studies on H. pylori infection. CONCLUSION: Gene expression data suggest that infection with H. pylori causes a decrease in glycan synthesis, resulting in shorter and simpler glycan structures.

Effects of combined progesterone and 17ß-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells.

A transcriptomic analysis of cultured human uterine smooth muscle cells (hUtSMCs) was performed to examine gene expression profiles in smooth muscle in an environment containing the two major steroid hormones that regulate the human myometrium in physiological states associated with estrous, pregnancy, labor, and pathophysiological states such as leiomyoma and endometrial cancer. hUtSMCs were treated with progesterone (P4) and 17ß-estradiol (E2) individually and in combination, in the presence and absence of RU486 (mifepristone). Transcription of many genes was modulated in the presence of P4 or E2 alone, but almost six times more genes were transcriptionally modulated in the presence of the P4/E2 hormone combination. In total 796 annotated genes were significantly differentially expressed in the presence of both P4 and E2 relative to their expression in untreated cells. Functional withdrawal of P4 by addition of RU486 effectively reversed almost all transcriptional changes caused by P4/E2 treatment. Gene ontology analysis of differentially expressed genes revealed a strong association between P4/E2 treatment and downregulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response, and differentiation. Upregulated processes included cell survival, gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signaling, and cell growth.

Transcriptomic effects of estradiol treatment on cultured human uterine smooth muscle cells.

Contractility of the myometrial smooth muscle cells during the estrous cycle and pregnancy is modulated by estrogen but the temporal expression of estrogen (relative to progesterone) and the type of contraction involved are distinctly different in pregnancy and estrous. This in vitro cell culture study investigated the global gene expression profile of human uterine smooth muscle cells (hUtSMCs) following 17ß-estradiol (E2) treatment. In response to E2 treatment 540 genes, many of which have not been previously described as estrogen responsive, were identified as significantly differentially expressed. These genes are involved in biological processes that include muscle contraction, cell migration and adhesion, apoptosis and phosphorylation. Evidence from this study suggests that 17ß-estradiol may have effects that are contrary to an overall contraction phenotype. The hUtSMC in vitro culture system is a useful model to investigate steroid effects on smooth muscle cells and may provide additional clues as to how smooth muscle cells behave in vivo.

Dietary vegetable oils do not alter the intestine transcriptome of gilthead sea bream (Sparus aurata), but modulate the transcriptomic response to infection with Enteromyxum leei.

BACKGROUND: Studies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes. RESULTS: Infection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated. CONCLUSIONS: The high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.

Functional analysis of muscle gene expression profiles associated with tenderness and intramuscular fat content in pork.

Warner-Bratzler shear force (WBSF) and % intramuscular fat content (IMF) are objective meat quality measurements that are significantly correlated with aspects of palatability such as tenderness, flavour and juiciness. Using cDNA microarrays, Musculus longissimus transcriptomic profiles at slaughter were compared in samples displaying lower or higher IMF (n=8) and WBSF values on day 1 post mortem (n=8). 101 identified genes were differentially expressed in relation to WBSF, while 160 genes were associated with differences in IMF. Reduced expression of protein synthesis genes and enhanced expression of genes involved in protein degradation were associated with lower WBSF values on day 1. Pathways including oxidative phosphorylation and the citrate cycle were significantly associated with higher IMF. Many lipid oxidation and fatty acid metabolism pathway genes were down-regulated in high IMF tissue, suggesting a suppression of fatty acid turnover in muscle with higher fat content. Identified genes provide targets for the discovery of novel genetic variation influential on pork palatability.

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress.

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.

Molecular profiling of the gilthead sea bream (Sparus aurata L.) response to chronic exposure to the myxosporean parasite Enteromyxum leei.

The aim of the present work was to investigate the transcriptome response of gilthead sea bream (Sparus aurata) after challenge with the myxosporean Enteromyxum leei, a wide-spread enteric parasite causing heavy economic losses in Mediterranean sparid farms. This parasite causes severe desquamative enteritis which usually leads to death of the fish, and there are no preventative or curative treatments for this enteromyxosis. After 113 days of exposure to parasite-contaminated effluent, fish were classified into three cohorts: control fish not exposed to parasite, those that were exposed and infected, and those that were exposed but not infected. In order to detect target genes that may be candidates for infective status or resistance, a cDNA microarray containing 18,490 cDNA clones enriched in genes differentially expressed after infection was hybridised with head kidney and intestine samples. In infected fish, 371 and 373 genes were differentially regulated at the >1.5-fold level in intestine and head kidney respectively, whereas in non-infected fish 175 and 501 genes were differentially regulated in these tissues, respectively. A global marked gene down-regulation was evident in infected fish, mainly in genes involved in the immune and acute phase response particularly complement and mannose binding lectin. Microarray analysis demonstrated a complex interplay between host and/or parasite derived proteases and protease inhibitors, apoptosis, cell proliferation and antioxidant defence genes in exposed fish. In the head kidney of non-infected fish a marked depression of genes involved in the acute phase response was evident. By contrast, in the intestine of non-infected fish, interferon-stimulated and MHC class II genes involved in antigen processing and presentation were up-regulated, possibly indicating that an active immune response at the local level is important to avoid infection with or proliferation of the parasite.

Use of microarray technology to assess the time course of liver stress response after confinement exposure in gilthead sea bream (Sparus aurata L.).

BACKGROUND: Selection programs for growth and stress traits in cultured fish are fundamental to the improvement of aquaculture production. The gilthead sea bream (Sparus aurata) is the main aquacultured species in the Mediterranean area and there is considerable interest in the genetic improvement of this species. With the aim of increasing the genomic resources in gilthead sea bream and identifying genes and mechanisms underlying the physiology of the stress response, we developed a cDNA microarray for gilthead sea bream that is enriched by suppression substractive hybridization with stress and immunorelevant genes. This microarray is used to analyze the dynamics of gilthead sea bream liver expression profile after confinement exposure. RESULTS: Groups of confined and control juvenile fish were sampled at 6, 24, 72 and 120 h post exposure. GeneSpring analyses identified 202 annotated genes that appeared differentially expressed at least at one sampling time (P < 0.05). Gene expression results were validated by quantitative PCR of 10 target genes, and K-means clustering of differently expressed genes identified four major temporal gene expression profiles. Set 1 encompassed a rapid metabolic readjustment with enhanced uptake and intracellular transport of fatty acids as metabolic fuels. Set 2 was associated with a wide variety of tissue repair and remodeling processes that were mostly mediated by the stress response of the endoplasmic reticulum (ER). Sets 3 and 4 encompassed the re-establishment of cellular homeostasis with increased intracellular trafficking and scavenging of reactive oxygen species (ROS), accompanied by a bidirectional regulation of the immune system and a general decline of ROS production. CONCLUSIONS: Collectively, these findings show the complex nature of the adaptive stress response with a clear indication that the ER is an important control point for homeostatic adjustments. The study also identifies metabolic pathways which could be analyzed in greater detail to provide new insights regarding the transcriptional regulation of the stress response in fish.

Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar).

BACKGROUND: The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult) and in the parr-smolt transformation. RESULTS: Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1), which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein) beta polypeptide 2-like 1 (GNB2L1) were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For anti-Mullerian hormone and guanine nucleotide binding protein beta polypeptide 2-like 1 expression changes in precocious males mirrored mature adults (November) but for collagen 1A and beta-globin the pattern was more complex. CONCLUSIONS: Expression changes in the fish brain during the process of precocious sexual maturation were small compared to those in the testes. Microarray analysis suggested down-regulation of housekeeping functions and up-regulation of a small number of specific processes. Transcriptional changes in the testes were much more pronounced with anti-Mullerian hormone playing a major role. Expression profiles for mature parr and maturing adult testes indicate subtle differences in gene expression between these two related groups.

Acute phase gene expression in rainbow trout (Oncorhynchus mykiss) after exposure to a confinement stressor: A comparison of pooled and individual data.

This study set out to investigate whether differential expression of genes for acute phase proteins in rainbow trout (Oncorhynchus mykiss) could be induced by confinement stress, a non-invasive method of activating the neuroendocrine stress response. In addition, a second objective was to assess the variation in gene expression between individual fish within the population of stressed fish in an attempt to identify APP genes having uniform and consistent changes in expression during stress. The liver was chosen for this investigation as it is the primary site of acute phase protein synthesis. Relative expression of the eight genes including transferrin, fibrinogen-like protein 2 (flp2), alpha-1-anti-proteinase-like protein (alpha1-antiprot), leukocyte cell-derived chemotaxin 2 (LECT2), pentraxin, serum amyloid A (SAA), haptoglobin (Hp), and differentially regulated trout protein 1 (DRTP1) was analysed by quantitative real-time PCR (qPCR) over 5 experimental time points spanning the course of a week. The results showed that the expression of three genes, SAA, haptoglobin and DRTP1, were most altered as a result of exposure to confinement stress. A correlation was identified between the expression of haptoglobin and DRTP1. Gene expression analyses in individual fish found that the transcript levels of haptoglobin and DRTP1 genes varied much less between individuals than was the case for SAA. The increase of haptoglobin and DRTP1 gene expression and its uniformity in response to stress make these genes potential biomarkers for stress in trout.

Gene expression and enzyme activity of mitochondrial proteins in irradiated rainbow trout (Oncorhynchus mykiss, Walbaum) tissues in vitro.

In recent years ethical, legislative and economic pressures have created a renewed interest in the development of alternatives to in vivo animal experiments. In vitro studies, particularly those using cell cultures, have been used increasingly as tools to assess the degree of toxicity associated with or present in particular environments. While cell cultures are useful to give relative toxicity values, genotypic and phenotypic integrity may be compromised in the continuous artificial environment they experience. In addition, cell cultures lack the complexity of functional organs and thus do not truly represent the effects that toxins exert on organ and organism functionality. In this study, ex vivo tissue cultures of rainbow trout gill, skin and spleen samples were analyzed for variation of expression in genes associated with oxidative phosphorylation after exposure to ionizing radiation. Significant radiation-induced changes in gene expression and enzyme activity associated with the mitochondrial oxidative phosphorylation process were identified. The tissues examined in this study demonstrated an exposure threshold at which radiation dose stimulates an alteration in the regulatory activity of mitochondrial-associated genes. Spleen tissues exposed to low levels of radiation (0.1 Gy) appeared most sensitive whereas skin tissues proved least sensitive, reacting only to higher doses (>1 Gy). We propose this investigative approach as an innovative alternative to in vivo studies because it identifies toxic exposure in vitro and could significantly reduce the number of live-animal toxicity tests required.

Characterisation of the differentially regulated trout protein 1 (DRTP1) gene in rainbow trout (Oncorhynchus mykiss).

Increased levels of differentially regulated trout protein 1 (DRTP1) mRNA transcripts have been reported in fish after activation of the acute phase response. While the function of the DRTP1 protein still remains to be elucidated, this study focused on the genomic organisation of the gene, the quantification of the DRTP1 transcript in various tissues, and the isolation and analysis of the 5' regulatory region of the DRTP1 gene in rainbow trout (Oncorhynchus mykiss). Analysis of the DRTP1 genomic and cDNA sequences showed the gene to consist of four exons separated by three introns. Tissue localisation of the DRTP1 gene was performed by Northern analysis and validated by quantitative real-time PCR (qPCR). Six tissues (liver, intestine, spleen, brain, pituitary, and hypothalamus) were analysed. The tissues with the most abundant transcripts were the liver and the pituitary, with lesser amounts detected in the intestine, hypothalamus, brain and spleen. Genome walking allowed the isolation of a 934 bp sequence of the 5' regulatory region of the gene which was cloned, sequenced and in which potential transcription factor binding sites were identified. Promoter fragments of decreasing size were generated and transiently transfected into the human hepatoma cell line (HepG2). Inducibility of the promoter was determined by stimulation of the HepG2 cells containing the constructs with dexamethasone, polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha). One construct, containing two potential C-EBP/beta sites and two NF-kappaB sites, exhibited the highest promoter induction (6.34 fold +/- SEM 0.5) when stimulated with human TNFalpha. A slightly shorter fragment containing one C-EBP/beta site and one NF-kappaB site did not show any significant inducibility when treated with TNFalpha. The loss of the C-EBP/beta and NF-kappaB in the shorter construct suggests that these sites, either individually or in combination, are critical for the induction of the DRTP1 promoter by TNFalpha.

The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

Cloning and characterization of two genes encoding rainbow trout homologues of the IFITM protein family.

To identify genes connected with leucocyte function in rainbow trout a cDNA library derived from head kidney leucocytes exposed to phytohemagglutinin (PHA) was screened. A clone with homology to the previously described human interferon-inducible transmembrane proteins (IFITM) was isolated. This clone was used to screen a trout genomic library identifying two trout IFITM genes. Both genes are composed of two exons, in common with human IFITM genes. The promoter regions of both genes were examined and found to contain interferon, immune and stress-related transcription factor binding sites. Transcripts for both genes were found in most tissues examined. Trout IFITM1 mRNA levels were highest in head kidney, but present in all tissues studied. IFITM2 mRNA levels were highest in gill, heart and liver, and absent in head kidney and blood. In response to conditions mimicking viral infection, transcription of the trout IFITM genes was elevated in a trout spleen macrophage cell line (RTS11) over the examined timecourse (72 h). This study suggests that the organization and transcription of the IFITM genes is well conserved between human and rainbow trout.

Isolation and characterization of the bovine Stearoyl-CoAdesaturase promoter and analysis of polymorphisms in the promoter region in dairy cows.

Conjugated linoleic acid (CLA) in milk arises through microbial biohydrogenation of dietary polyunsaturated fatty acids (PUFA) in the rumen, and by the action of mammary Stearoyl-CoA desaturase (Scd). A large variation (up to tenfold) in the concentration of this fatty acid in milk has been observed, even in cows receiving the same diet. The reasons for this variation are not well understood. In this study the bovine core promoter region was isolated by a genome walking strategy from genomic DNA Genome Walker libraries and then cloned and characterized. This core promoter sequence extended approximately 600 bp upstream of the translation start site. The presence of putative transcription factor binding sites conserved in bovine, human, and mouse promoters was observed. Evidence that this promoter fragment was functional in vivo was obtained from expression studies in a mammary cell line. The promoter sequence of the scd gene was compared between cows selected for the ability to produce high fatty acid methyl esters (FAME) (2.22-2.72) in their milk, with the same promoter region of low-FAME-producing cows (0.81-1.12). However, such comparisons of the sequences of the scd promoter region of cows producing high milk CLA compared with low CLA revealed no polymorphisms in this promoter segment. Furthermore, no sequence polymorphisms were observed among the scd promoter region of Holstein Friesian, Montbeliarde, Normande, Norwegian Red, Charlois, Limousin and Kerry breeds.