The healthy equine uterus harbors a distinct core microbiome plus a rich and diverse microbiome that varies with geographical location.

The goal of this study was to understand the composition and existence of the resident uterine microbiome in healthy mares and to establish the presence of a core microbiome for the healthy equine uterus. We analyzed the microbiomes of 35 healthy mares that are long-time residents of three farms in Oklahoma, Louisiana, and Australia as well as that of 19 mares purchased from scattered owners in the Southern Mid-Western states of the United States. Over 6 million paired-end reads of the V4 region of the 16S rRNA gene were obtained resulting in 19,542 unique Amplicon Sequence Variants (ASVs). ASVs were assigned to 17 known phyla and 213 known genera. Most abundant genera across all animals were Pseudomonas (27%) followed by Lonsdalea (8%), Lactobacillus (7.5%), Escherichia/Shigella (4.5%), and Prevotella (3%). Oklahoma and Louisiana samples were dominated by Pseudomonas (75%). Lonsdalea (28%) was the most abundant genus in the Australian samples but was not found in any other region. Microbial diversity, richness, and evenness of the equine uterine microbiome is largely dependent on the geographical location of the animal. However, we observed a core uterine microbiome consisting of Lactobacillus, Escherichia/Shigella, Streptococcus, Blautia, Staphylococcus, Klebsiella, Acinetobacter, and Peptoanaerobacter.

Canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems.

The objective of this study was to characterize the normal microbiome of healthy canine vagina and endometrium and to determine the effect of the stage of estrous, on the resident microbiome. Cranial vaginal swabs and uterine biopsy samples were collected from twenty-five bitches in five different stages of estrous at elective ovariohysterectomy (OVH). Over 4 million reads of the V4 region of 16S rDNA gene were obtained and used for further analyses. A total of 317 genera belonging to 24 known phyla were identified. The endometrium was higher in bacterial diversity while the vagina was higher in richness. Proteobacteria, Bacteroidetes and Firmicutes were the most abundant phyla observed across all samples. Hydrotalea, Ralstonia, and Fusobacterium accounted for nearly 60% of the OTUs identified in the vagina while organisms identified in the endometrium were more evenly distributed. Pseudomonas, Staphylococcus, and Corynebacterium were the prominent genera in the endometrium. The microbiome of the endometrium was distinctly different from that of the vagina. There was large animal-to-animal variation. Other than the vaginal microbiome of bitches in estrus (i.e. in heat), there were no distinct clustering of the organisms based on the stage of estrous. These findings establish the presence of a resident microbiome of the endometrium throughout all stages of estrous cycle.

Technical note: Validation of an automated system for monitoring and restricting water intake in group-housed beef steers.

The Insentec Roughage Intake Control (RIC) system has been validated for the collection of water intake; however, this system has not been validated for water restriction. The objective of this validation was to evaluate the agreement between direct observations and automated intakes collected by the RIC system under both ad libitum and restricted water conditions. A total of 239 crossbred steers were used in a 3-d validation trial, which assessed intake values generated by the RIC electronic intake monitoring system for both ad libitum water intake ( = 122; BASE) and restricted water intake ( = 117; RES). Direct human observations were collected on 4 Insentec water bins for three 24-h periods and three 12-h periods for BASE and RES, respectively. An intake event was noted by the observer when the electronic identification of the animal was read by the transponder and the gate lowered, and starting and ending bin weights were recorded for each intake event. Data from direct observations across each validation period were compared to automated observations generated from the RIC system. Missing beginning or ending weight values for visual observations occasionally occurred due to the observer being unable to capture the value before the monitor changed when bin activity was high. To estimate the impact of these missing values, analyses denoted as OBS were completed with the incomplete record coded as missing data. These analyses were contrasted with analyses where observations with a single missing beginning or end weight (but not both) were assumed to be identical to that which was recorded by the Insentec system (OBS). Difference in mean total intake across BASE steers was 0.60 ± 2.06 kg OBS (0.54 ± 1.99 kg OBS) greater for system observations than visual observations. The comparison of mean total intake across the 3 RES validation days was 0.53 ± 2.30 kg OBS (0.13 ± 1.83 kg OBS) greater for system observations than direct observations. Day was not a significant source of error in this study ( > 0.05). These results indicate that the system was capable of limiting water of individual animals with reasonable accuracy, although errors are slightly higher during water restriction than during ad libitum access. The Insentec system is a suitable resource for monitoring individual water intake of growing, group-housed steers under ad libitum and restricted water conditions.

Coordinated gene expression between skeletal muscle and intramuscular adipose tissue in growing beef cattle.

Previous research indicates that metabolism and fiber type of skeletal muscle is related to intramuscular lipid content. It is hypothesized that changes in skeletal muscle gene expression influence adipose tissue development. The objective of this study was to determine differences in the metabolism and intercellular signaling of skeletal muscle fibers within the same muscle group that could be responsible for the initiation of intramuscular adipose tissue development and differentiation. Longissimus dorsi muscle samples were collected from steers ( = 12; 385 d of age; 378 kg BW) grazing wheat pasture. Longissimus muscle samples were dissected under magnification and sorted into 3 categories based on visual stage of adipose tissue development: immature intramuscular adipose tissue (MM), intermediate intramuscular adipose tissue (ME), and mature intramuscular adipose tissue (MA). Additionally, muscle fibers lying adjacent to each intramuscular adipose tissue (IM) category and those not associated with IM tissue were collected and stored separately. Quantitative real-time PCR was used to determine relative fold change in genes involved in metabolism, angiogenesis, formation of extracellular matrix, and intercellular signaling pathways in both LM and IM samples. Gene expression data were analyzed using a GLM that included the fixed effect of tissue. Pearson correlation coefficients were also computed between gene expression in LM and IM tissue samples that were at the same stage of development. and γ mRNA expression were 3.56- and 1.97-fold greater ( < 0.05) in ME and MA IM compared with MM IM whereas mRNA expression was 1.43-fold less ( < 0.01) in MA IM compared with MM IM, indicating successful separation into different development categories. Genes associated with metabolism and angiogenesis in LM tissue showed no differences among stages of development. Myostatin expression did not change in LM tissue; however, expression of and mRNA decreased ( < 0.01) as IM matured. and mRNA expression were 2.5- and 1.32-fold greater in LM associated with MM IM than in LM associated with ME IM. Angiogenic growth factors in MM IM tissue had a strong positive correlation ( ≥ 0.69) with angiogenic growth factors in LM associated with MM IM; however, no correlation was observed in ME or MA IM. These data indicate a coordinated effort between LM and IM in early stages of IM development.

Effects of starch- vs. fiber-based energy supplements during winter grazing on partitioning of fat among depots and adipose tissue gene expression in growing cattle and final carcass characteristics.

Fifty-five normal-weaned Angus steers (268 ± 22 kg; 265 ± 16 d of age) were used to evaluate the effects of starch- vs. fiber-based energy supplements for stocker cattle grazing low-quality dormant native range on growth performance, body composition, and adipose tissue development of different fat depots. Steers were randomly allotted to 4 treatments: 1.02 kg·steer(-1)·d(-1) of a 40% CP cottonseed meal-based supplement (CON), corn/soybean meal-based supplement fed at 1% of BW (CORN), soybean hull/soybean meal-based supplement fed at 1% of BW (SBH), or dried distillers grains with solubles fed at 1% of BW (DDGS). All supplements were individually fed 5 d/wk during the 121-d winter grazing phase. After winter grazing, 3 steers per treatment were harvested to determine body composition and carcass characteristics, and collect subcutaneous (SC) and perirenal (PR) adipose tissue samples. The remaining steers grazed cool-season grass pastures for 74 d without supplementation before finishing. Steers were fed a common finishing diet for 113 d before harvest, at which time carcass characteristics were collected at a commercial abattoir. Energy supplementation increased (P < 0.01) winter grazing ADG compared with CON steers, and CORN steers had greater (P < 0.01) ADG than SBH and DDGS steers. Energy supplementation increased (P < 0.04) mesenteric/omental fat mass but did not influence (P > 0.13) 12th rib fat thickness or marbling score at intermediate harvest compared with CON steers. The mRNA expression of genes involved in lipogenesis and markers of adipogenesis were greater (P < 0.05) in PR adipose tissue of energy-supplemented steers compared with CON steers but not in SC adipose tissue. Fiber-supplemented steers had greater (P < 0.01) mRNA expression of fatty acid synthase and fatty acid binding protein 4 compared with CORN steers in PR adipose tissue but not SC adipose tissue. At final harvest, energy-supplemented steers had greater (P < 0.05) KPH and yield grade than CON steers, but no differences (P = 0.75) in marbling score were observed. Neither energy supplementation nor type of energy supplement influenced intramuscular fat deposition in stocker cattle grazing dormant native range. These data suggest that the total energy intake and stage of animal maturity during grazing supplementation were not great enough to influence marbling deposition.

Rumen microbial population dynamics during adaptation to a high-grain diet.

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.

Effects of nutrient restriction of bovine dams during early gestation on postnatal growth, carcass and organ characteristics, and gene expression in adipose tissue and muscle.

Angus x Hereford heifers (15 mo and artificially inseminated to a single sire) were used to evaluate the effect of prenatal nutritional restriction on postnatal growth and development. At d 32 of gestation, dams were stratified by BW and BCS and allotted to a low-nutrition [55% of NRC (1996) requirements, n = 10] or moderate-nutrition [100% of NRC (1996) requirements, n = 10] diet. After 83 d of feeding, dams were commingled and received a diet in excess of requirements. Dams were allowed to calve naturally, and birth weights and growth of calves were recorded. Bulls were castrated at birth. Steers (16 mo of age, 5 per treatment) received a high-concentrate diet ad libitum to a constant age (88 ± 1 wk). Steers were slaughtered and weights of the empty body and organs were recorded. Samples of organs, muscle (complexus), and perirenal and subcutaneous adipose tissue were stored at -80 degrees C, and then DNA and protein concentrations were quantified and expression of genes associated with fatty acid metabolism and glucose uptake were measured in adipose and muscle tissue. Dams had similar (P > 0.33) BW and BCS at the beginning of the experiment. At the end of restriction, dams on the low-nutrition diet weighed less (P ≤ 0.01) and had less BCS (P < 0.001) than those on the moderate-nutrition diet. Length of gestation was 274 ± 2 d for dams in the low-nutrition treatment and 278 ± 2 d (P = 0.05) for dams in the moderate-nutrition treatment. Nutrient restriction during gestation did not influence birth weight or postnatal growth of calves. Lungs and trachea of steers whose dams were fed the low-nutrition diet weighed less (P = 0.05) at slaughter than those of steers whose dams were fed the moderate-nutrition diet; weights of other organs were not influenced by treatment. Complexus muscle from steers whose dams were fed the low-nutrition diet had a greater (P = 0.04) concentration of DNA and larger muscle fiber area compared with steers whose dams were fed the moderate-nutrition diet. Abundance of mRNA for fatty acid binding protein 4, fatty acid translocase, and glucose transporter 4 was less in perirenal adipose tissue of steers whose dams were fed the low-nutrition diet compared with those whose dams were fed the moderate-nutrition diet. Nutritional restriction of dams during early gestation did not alter postnatal calf growth. However, concentrations of DNA in muscle tissue and muscle fiber area were greater in steers from dams exposed to restricted nutrient intake during early gestation.

Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies (Diptera: Muscidae).

The recent outbreak of Escherichia coli O157:H7 infection associated with contaminated spinach led to an investigation of the role of insects, which frequent fields of leafy greens and neighboring rangeland habitats, in produce contamination. Four leafy greens fields adjacent to cattle-occupied rangeland habitats were sampled using sweep nets and sticky traps. Agromyzid flies, anthomyiid flies, and leafhoppers were caught consistently in both rangeland and leafy greens production fields at all sites. An unexpected number of flies (n = 34) in the Muscidae and Calliphoridae families (known as filth flies because of their development in animal feces) were caught in one leafy greens field. A subset of these filth flies were positive (11 of 18 flies) for E. coli O157:H7 by PCR amplification using primers for the E. coli O157:H7-specific eae gene. Under laboratory conditions, house flies were confined on manure or agar medium containing E. coli O157:H7 tagged with green fluorescent protein (GFP) and then tested for their capacity to transfer the microbes to spinach plants. GFP-tagged bacteria were detected on surfaces of 50 to 100% of leaves examined by fluorescence microscopy and in 100% of samples tested by PCR. These results indicate that flies are capable of contaminating leafy greens under experimental conditions and confirm the importance of further investigation of the role of insects in contamination of fresh produce.

Porcine kallikrein gene family: genomic structure, mapping, and differential expression analysis.

Kallikreins belong to a family of serine proteases that are widespread throughout living organisms, expressed in diverse tissue-specific patterns, and known to have highly diverse physiological functions. The 15 human and 24 mouse kallikreins have been implicated in pathophysiology of brain, kidney, and respiratory and reproductive systems and often are used as cancer biomarkers. To better elucidate the structure and evolutionary origin of this important gene family in the pig, we have constructed a contiguous BAC clone-derived physical map of the porcine kallikrein gene region and have fully sequenced a BAC clone containing 13 kallikrein genes, 11 of which are novel. Radiation hybrid mapping assigns this kallikrein-gene-rich region to porcine chromosome 6. Phylogenetic and percent identity plot-based analyses revealed strong structure and order conservation of kallikreins among four mammalian species. Reverse transcriptase-polymerase chain reaction-based expression analysis of porcine kallikreins showed a complex expression pattern across different tissues with the thymus being the only tissue expressing all 13 kallikrein genes. [The sequence data described in this paper has been submitted to GenBank under Accession No. AC149292].

Porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 gene expression.

Previous studies have suggested that the porcine endometrium may express several tissue kallikreins during the estrous cycle and early pregnancy. The present study investigated porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 mRNA expression during the estrous cycle and early pregnancy. Tissue kallikrein (KLK) gene expression was evaluated using quantitative RT-PCR and in situ hybridization. KLK1 expression was similar across the estrous cycle and early pregnancy, and localized to the endometrial luminal (L) and glandular (G) epithelium. KLK4 endometrial mRNA expression was greatest on days 0, 5, and 10 when compared with days 12, 15, and 17 of the estrous cycle and greater in cyclic compared with pregnant gilts. Expression of KLK4 was more intense in the stroma and uterine epithelium from days 0 to 10 of the estrous cycle. Endometrial KLK11 mRNA was not different between cyclic and pregnant gilts but the expression was greatest on days 10 and 12 compared with all other days evaluated. There was an increased intensity of KLK11 gene expression in the stratum compactum on day 10 of the estrous cycle and early pregnancy. Endometrial KLK14 mRNA expression was not detectable on days 5 and 10 but was expressed on days 0, 12, 15, and 17 of the estrous cycle and pregnancy. KLK14 expression was localized in the uterine L and G epithelium, and stroma throughout the endometrium after day 10. Conceptus KLK1 mRNA did not change from days 10 to 17 of gestation. However, conceptus KLK4, and 14 mRNA expression was greatest on day 10 with expression declining after day 14 of gestation. Expression of the various tissue kallikreins in the endometrium and conceptus during the estrous cycle and early pregnancy in the pig can serve in the activation of growth factors and tissue remodeling during the establishment of pregnancy.

Maternal recognition of pregnancy signal or endocrine disruptor: the two faces of oestrogen during establishment of pregnancy in the pig.

Timing of conceptus growth and attachment to the uterine luminal epithelium is regulated by progesterone secretion from the corpus luteum and by expression of progesterone receptor in the uterine epithelia and stroma. Conceptus growth and uterine attachment are temporally associated with the disappearance of progesterone receptors from uterine epithelia. While the loss of progesterone receptor from the endometrial epithelia on day 10 of the oestrous cycle and pregnancy has been well documented, the factors involved with cell specific down-regulation of progesterone receptor are yet to be established. We propose that several progesterone stimulated factors activate nuclear factor kappa B (NF-kB) within the uterine epithelia, which leads to inhibition of progesterone receptor and concomitant stimulation of endometrial genes expressed during early conceptus development. Although oestrogens secreted by pig conceptuses function to establish pregnancy, timing of endometrial exposure to oestrogen is critical. Early oestrogen administration alters the pattern of gene expression through the NF-kB system desynchronising the uterine environment for conceptus implantation resulting in later embryonic loss.

Effects of grazing program and subsequent finishing on gene expression in different adipose tissue depots in beef steers.

This experiment was conducted to examine the effects of grazing program and subsequent finishing on gene expression in adipose tissue from steers. Twenty Angus x Angus-Hereford steer calves (initial BW = 231 +/- 25 kg) were allotted randomly to one of two winter grazing treatments: 1) grazing winter wheat pasture to achieve a high rate of BW gain (HGW); or 2) grazing dormant tallgrass native range (NR). Steers in the NR treatment were provided 0.91 kg.steer(-1).d(-1) of a 41% CP (as-fed basis) cottonseed meal supplement. Following the grazing period, steers were assigned randomly to feedlot pens. Steers were fed to a common endpoint of 1.27 cm of backfat between the 12th and 13th rib. Four steers from each treatment were slaughtered at the end of the grazing period, and the remaining steers from each treatment (n = 6) were slaughtered at the predetermined compositional endpoint. Intramuscular and s.c. fat samples were collected from LM sections of each steer at the 12th-/13th-rib interface on the left side. Pools of RNA were prepared for HGW and NR s.c. adipose tissue from steers slaughtered immediately after grazing. Suppression subtractive hybridization was performed followed by dot-blot hybridization screening to confirm differential expression of subtracted transcripts. Transcripts confirmed to be differentially expressed were subjected to dideoxy chain-termination sequencing. Quantitative reverse transcription PCR was performed on three differentially expressed clones: osteonectin, ferritin heavy chain, and decorin. Osteonectin, ferritin heavy chain, and decorin gene expression was greater (P < 0.05) in s.c. than in i.m. adipose tissue of finished steers. A depot x background interaction for osteonectin (P < 0.01) and ferritin heavy chain (P = 0.03) gene expression was observed for steers slaughtered after grazing, indicating that nutritional management can affect gene expression in adipose tissue depots differently. No differences resulting from prefinishing nutritional background (HGW or NR) were noted in osteonectin, ferritin heavy chain, or decorin gene expression in i.m. adipose tissue collected from finished steers, which might have resulted from feeding steers to the same compositional endpoint. Our data suggest that nutritional background alters gene expression in adipose depots, and that depots are influenced differently.

Gene expression in sheep skin and wool (hair).

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10(-4). In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin.

Porcine endometrial expression of kininogen, factor XII, and plasma kallikrein in cyclic and pregnant gilts.

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.

Systematic screening of sheep skin cDNA libraries for microsatellite sequences.

65,000 sheep skin cDNA clones were gridded in high density on to nylon membranes and screened for (CA)n and (GA)n repeat containing clones. 296 dinucleotide repeat-containing clones were identified with approximately 85% non-redundancy. Clones were single-pass 5' sequenced and we compared the Expressed Sequence Tag (EST) sequences to the Swiss-Prot database to ascertain their identity and/or putative function. We then aligned the ESTs against the human genomic sequence to determine the locations of human orthologous sequences. Finally, we developed a subset of polymorphic microsatellite markers and positioned them on the ovine linkage map.

Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep.

1,144 sheep belonging to 21 breeds and known crosses were sequence analyzed for polymorphisms in the ovine PRNP gene. Genotype and allele frequencies of polymorphisms in PRNP known to confer resistance to scrapie, a fatal neurodegenerative disease of sheep, are reported. Known polymorphisms at codons 136 (A/V), 154 (H/R) and 171 (Q/R/H/K) were identified. The frequency of the 171R allele known to confer resistance to type C scrapie was 53.8% and the frequency of the 136A allele known to influence the resistance to type A scrapie was 96.01%. In addition, we report the identification of five new polymorphisms at codons 143 (H/R), 167 (R/S), 180 (H/Y), 195 (T/S) and 196 (T/S). We also report the identification of a novel allele (S/R) at codon 138.

Divergent human and mouse orthologs of a novel gene (WBSCR15/Wbscr15) reside within the genomic interval commonly deleted in Williams syndrome.

Williams syndrome (WS) is a contiguous gene deletion disorder resulting in complex and intriguing clinical features. Detailed molecular characterization studies of the genomic segment on human chromosome 7q11.23 commonly deleted in WS have uncovered numerous genes, each of which is being actively studied for its possible role in the etiology of the syndrome. Our efforts have focused on the comparative mapping and sequencing of the WS region in human and mouse. In previous studies, we uncovered important differences in the long-range organization of these human and mouse genomic regions; in particular, the notable absence of large duplicated blocks of DNA in mouse that are present in human. Aided by available genomic sequence data, we have used a combination of gene-prediction programs and cDNA isolation to identify the human and mouse orthologs of a novel gene (WBSCR15 and Wbscr15, respectively) residing within the genomic segment commonly deleted in WS. Unlike the flanking genes, which are closely related in human and mouse, WBSCR15 and Wbscr15 are strikingly different with respect to their cDNA and corresponding protein sequences as well as tissue-expression pattern. Neither the WBSCR15- nor Wbscr15-encoded amino acid sequence shows a statistically significant similarity to any characterized protein. These findings reveal another interesting evolutionary difference between the human and mouse WS regions and provide an additional candidate gene to evaluate with respect to its possible role in the pathogenesis of WS.

Molecular characterization of the mouse p47-phox (Ncf1) gene and comparative analysis of the mouse p47-phox (Ncf1) gene to the human NCF1 gene.

The cytosolic factor p47-phox (NCF1) is a key component of the phagocyte NADPH-oxidase system, critical for microbicidal activity. The human p47-phox gene has been well characterized and resides on chromosome 7q11. Here we describe the molecular characterization of the mouse ortholog (Ncf1), which maps to distal chromosome 5, and compare the structure of the genes, commenting on the degree of homology. The mouse and human genes contain the same number of exons and introns, but the mouse gene is more compact (7.8 kb versus 15.2 kb). A percentage identity plot analysis comparing the human and mouse genes indicates that sequence homology is generally restricted to exons and does not include any large segment of introns or the 5' flanking sequence. The mouse gene also contains notably fewer repetitive elements than its human counterpart (34% versus 50%). The start of transcription of the mouse gene has been localized to within 12 nucleotides of the translation start site, similar to the human ortholog. Our findings provide an important foundation for investigating the evolutionary history of the p47-phox gene, particularly as it relates to understanding the molecular basis of the p47-phox-deficient autosomal recessive form of chronic granulomatous disease.

Comparative mapping of the region of human chromosome 7 deleted in williams syndrome.

Williams syndrome (WS) is a complex developmental disorder resulting from the deletion of a large (approximately 1.5-2 Mb) segment of human chromosome 7q11.23. Physical mapping studies have revealed that this deleted region, which contains a number of known genes, is flanked by several large, nearly identical blocks of DNA. The presence of such highly related DNA segments in close physical proximity to one another has hampered efforts to elucidate the precise long-range organization of this segment of chromosome 7. To gain insight about the structure and evolutionary origins of this important and complex genomic region, we have constructed a fully contiguous bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) contig map encompassing the corresponding region on mouse chromosome 5. In contrast to the difficulties encountered in constructing a clone-based physical map of the human WS region, the BAC/PAC-based map of the mouse WS region was straightforward to construct, with no evidence of large duplicated segments, such as those encountered in the human WS region. To confirm this difference, representative human and mouse BACs were used as probes for performing fluorescence in situ hybridization (FISH) to metaphase and interphase chromosomes. Human BACs derived from the nonunique portion of the WS region hybridized to multiple, closely spaced regions on human chromosome 7q11.23. In contrast, corresponding mouse BACs hybridized to a single site on mouse chromosome 5. Furthermore, FISH analysis revealed the presence of duplicated segments within the WS region of various nonhuman primates (chimpanzee, gorilla, orangutan, and gibbon). Hybridization was also noted at the genomic locations corresponding to human chromosome 7p22 and 7q22 in human, chimpanzee, and gorilla, but not in the other animal species examined. Together, these results indicate that the WS region is associated with large, duplicated blocks of DNA on human chromosome 7q11.23 as well as the corresponding genomic regions of other nonhuman primates. However, such duplications are not present in the mouse.