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Summary In a previous study two bulls were identified in the Polish Black-and-White Lowland cattle population that inherited an increased serum lytic activity (LZM+ ) in a heterozygous fashion. The LZM+ phenotype shows in both half-sib families a strong co-segregation with a particular allele of the LysMic microsatellite localized within intron 2 of the immunorelevant macrophage expressed lysozyme encoding-gene (mLYZ ). No trait-associated mLYZ promoter variant had previously been found. Here, we examine genetic polymorphisms within the entire coding region of this gene by comparative sequence analyses of all four exons in both bulls and two progenies exhibiting low serum lytic activity (LZM0 ). We found no mutations in the coding sequence of mLYZ gene. However, two single nucleotide polymorphisms were detected in introns 2 and 3 of this mLYZ gene at positions 8603 (C/T transition) and 9963 (C/A transversion). A polymerase chain reaction-restriction fragment length polymorphism assay was developed to detect the C/T transition in intron 2, creating a polymorphic Sau 3A restriction site. The ‘T’-residue-harbouring allele segregates with the LZM+ phenotype and is tightly linked to the previously reported diagnostic allele 7 of the LysMic microsatellite. Unaltered DNA sequences in both, promoter and coding region of mLYZ encoding gene in animals with high and low lytic activity indicates that this parameter is influenced by factors other than quantity or quality of the enzyme encoded by this gene. [ABSTRACT FROM AUTHOR]

Performance of biological functions of lactoferrin in the small intestine requires at least some resistance to degradation. Therefore, we studied prececal digestibility of lactoferrin in comparison to casein both in suckling and adult miniature pigs, applying 15N-labeled proteins. In study 1, 43 piglets (10-d-old), deprived of food for 12 h received 10 mL of sow's milk supplemented with 120 mg of 15N-labeled protein (porcine or bovine lactoferrin or bovine casein). Piglets were anesthetized 150 min later, after which the small intestine was excised, cut into three sections, and chyme was collected. In study 2, nine food-deprived boars fitted with T-canulae at the terminal ileum were given two semisynthetic experimental meals (204 g) in a cross-over design, 2 wk apart. One contained 7.5% (g/100 g) 15N-labeled bovine casein, the other 1.25% 15N-labeled bovine lactoferrin. Both were adjusted to 15% total protein with nonlabeled casein. Ileal chyme was collected from the canula over 33 h postprandially. All diets contained the indigestible marker chromic oxide. 15N-digestibility of lactoferrin, both porcine (84.4 +/- 3.2%) and bovine (82.3 +/- 4.8%), was significantly lower than casein digestibility (97.6 +/- 0.5%) in the distal small intestine of suckling piglets (P < 0.05). Based on immunoblotting after acrylamide electrophoresis, 4.5% of non- and partially digested lactoferrin was found in the last third of the small intestine of piglets. In adult miniature pigs there was no difference in 15N-digestibility of bovine lactoferrin compared to bovine casein (90.7 +/- 1.9% vs. 93.9 +/- 1.0%, P > 0.05). In suckling miniature pigs, the reduced digestibility of lactoferrin may provide the prerequisite for biological actions along the whole intestinal tract. The source of lactoferrin, porcine or bovine, made no difference in this respect. [ABSTRACT FROM AUTHOR]

We present the echocardiographic images of a patient with transient ischaemic attack with double atrial septum and persistent interatrial space as probable source of thrombus. A patent foramen ovale could be excluded and a communication between left atrium and interatrial space could be demonstrated. [ABSTRACT FROM PUBLISHER]

Expression of Hp mRNA, one major acute phase protein in cattle, was detected in homogenates of the bovine mammary gland. The aim of this study was to localize Hp mRNA expression within the udder at the cellular level and to quantify any difference of the level of this expression caused by two major mastitis pathogens. For this purpose 3 quarters of each of 3 cows were subsequently inoculated with Escherichia coli (E. coli) at 6, 12 and 24 h pre-slaughter, the 4th quarter received saline 24 h pre-slaughter as control. Another 3 cows received Staphylococcus aureus (S. aureus) accordingly. After slaughter, tissue samples of each quarter were collected for analyses by in situ hybridization (ISH) and real-time RT-PCR. ISH allocated Hp mRNA expression to the alveolar epithelium of the mammary gland. In addition, evaluating the number of cells expressing Hp mRNA revealed a significant difference between the 4 quarters treated with E. coli (P<0.05) in contrast to the quarters exposed to S. aureus. A 17-fold rise in the number of epithelial cells expressing Hp in mammary tissues was detected during the course of E. coli infection, whereas Hp mRNA expression in response to S. aureus infection appeared unchanged. This divergence was confirmed by quantitative real-time RT-PCR: in contrast to S. aureus infected animals, Hp mRNA expression differed significantly between the quarters of E. coli infected animals (P<0.05). In the latter a 176-fold increase was observed from 0 to 24 h. In summary, this is the first study reporting the localization of Hp mRNA in mammary epithelium of cows. The lack of response to S. aureus within the first 24 h after infection is in contrast to E. coli infection and possibly highlights pathogen-species dependent modulation of the host's immune defense in the udder [ABSTRACT FROM AUTHOR]

Examines the localization of STAT3-encoding gene near STAT5A and STAT5B-encoding genes in cattles. Factors involved in the regulation of immune response during acute-phase-response following infection; Function of prolactin signals during lactation cycle; Mediators of growth hormone signaling.

Diets containing corn starch may improve glucose supply by providing significant amounts of intestinal starch and increasing intestinal glucose absorption in dairy cows. Glucose absorption in the small intestine requires specific glucose transporters; that is, sodium-dependent glucose co-transporter-1 (SGLT1) and facilitated glucose transporter (GLUT2), which are usually downregulated in the small intestine of functional ruminants but are upregulated when luminal glucose is available. We tested the hypothesis that mRNA and protein expression of intestinal glucose transporters and mRNA expression of enzymes related to gluconeogenesis are affected by variable starch supply. Dairy cows (n = 9/group) were fed for 4 wk total mixed rations (TMR) containing either high (HS) or low (LS) starch levels in the diet. Feed intake and milk yield were measured daily. After slaughter, tissue samples of the small intestinal mucosa (mid-duodenum and mid-jejunum) were taken for determination of mRNA concentrations of SGLT1 and GLUT2 as well as pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase by real-time reverse transcription PCR relative to a housekeeping gene. Protein expression of GLUT2 in crude mucosal membranes and of SGLT1 and GLUT2 in brush-border membrane vesicles was quantified by sodium dodecyl sulfate-PAGE and immunoblot. A mixed model was used to examine feeding and time-related changes on feed intake and milk yield and to test feeding and gut site effects on gene or protein expression of glucose transporters and enzymes in the intestinal mucosa. Dry matter intake, but not energy intake, was higher in cows fed HS compared with LS. Abundance of SGLT1 mRNA tended to be higher in duodenal than in jejunal mucosa, and mRNA abundances of pyruvate carboxylase tended to be higher in jejunal than in duodenal mucosa. In brush-border membrane vesicles, SGLT1 and GLUT2 protein expression could be demonstrated. No diet-dependent differences were found concerning mRNA and protein contents of glucose transporter or mRNA level of gluconeogenic enzymes. In conclusion, our investigations on glucose transporters and gluconeogenic enzymes in the small intestinal mucosa of dairy cows did not show significant diet regulation when TMR with different amounts of intestinal starch were fed. Therefore, predicted intestinal glucose absorption after enhanced starch feeding is probably not supported by changes of intestinal glucose transporters in dairy cows. [ABSTRACT FROM AUTHOR]

Effects of dietary fat supplementation prepartum on liver lipids and metabolism in dairy cows are contradictory. Thus, we examined in 18 German Holstein cows (half-sib; first lactation 305-d milk yield >9,000 kg) whether dietary fat:carbohydrate ratio during the last trimester of gestation affects lipid metabolism and milk yield. The diets were formulated to be isoenergetic and isonitrogenous but differed in rumen-protected fat (FD; 28 and 46.5 g/kg of dry matter during far-off and close-up dry period; mainly C16:0 and C18:1) and starch concentration [carbohydrate diet (CD); 2.3 times as much starch as FD]. Diets were given ad libitum starting 12 wk before expected parturition. After parturition all cows were fed a single lactation diet ad libitum for 14 wk. With the FD treatment, dry matter intake was depressed prepartum, milk yield during first 4 wk of lactation was lower (36.9 vs. 41.0 kg/d), and postpartum energy balance during this period was more negative. During the first 4 wk, cows in the FD group had lower lactose percentage and yield but higher milk fat, whereas milk protein and fat yield as well as energy-corrected milk did not differ. Between wk 5 and 14, milk fat and milk protein percentage was lower in CD than in FD. Milk fat C14:0 was lower and C16:1 was higher in the FD group. For FD cows, plasma triacylglycerol, nonesterified fatty acids, and cholesterol concentrations were higher prepartum, whereas plasma β-hydroxybutyrate and glucose concentrations were lower. During the first 10 d after parturition, plasma triacylglycerol concentration was higher in FD, and prepartum plasma glucose and cholesterol differences persisted during the first 14 wk of lactation. Irrespective of prepartum nutrient composition, concentrations of plasma leptin and subcutaneous fat leptin mRNA decreased between -10 d to +10 d relative to parturition, and liver lipids and glycogen reached maximum and minimal values, respectively, 10 d after parturition. Acetyl-coenzyme A carboxylase α mRNA abundance in subcutaneous fat decreased between -10 d to +1 d relative to parturition by 97%, whereas it was generally much lower in the liver and remained at a low level until wk 14 of lactation. In conclusion, feeding a diet containing rumen-protected fat during late lactation and dry period until calving negatively affected dry matter intake, energy balance, and milk yield during subsequent lactation, did not change acetyl-coenzyme A carboxylase α mRNA abundance in subcutaneous fat, and was not beneficial for liver lipid accumulation. [ABSTRACT FROM AUTHOR]

Expression of haptoglobin (Hp) mRNA, one of the major acute phase proteins in cattle, was demonstrated in homogenates of the bovine mammary gland. The aim of this study was to localize Hp mRNA expression within the udder at the cellular level during the first 24 h of infection with Escherichia coli. For this purpose, 3 quarters of 3 cows were subsequently inoculated with E. coli at 6, 12, and 24 h preslaughter; the fourth quarter received saline at 24 h preslaughter as a control. After slaughter, tissue samples of each quarter were collected for analyses by in situ hybridization and real-time reverse-transcription PCR. Haptoglobin mRNA expression was allocated to the alveolar epithelium of the mammary gland. Quantification of lip-positive cells in in situ hybridization of lip mRNA from tissue homogenates and of Hp protein in milk confirmed increasing concentrations within 24 h of infection. [ABSTRACT FROM AUTHOR]

Subacute ruminal acidosis (SARA) of dairy cattle is a widely occurring but not very overt metabolic disorder thought to impair milk composition. The enzyme stearoyl-CoA desaturase 1 (SCD1) is rate-limiting for the formation of Δ-9 unsaturated fatty acids and thus crucially involved in controlling lipid metabolism in the liver. It is known that SCD1 expression is downregulated during SARA, but the underlying molecular mechanisms are unknown. To study these mechanisms, we enrolled 12 healthy multiparous mid-lactation Holstein cows into a diet-induced SARA experiment. Six cows were fed a high-concentrate diet for 18 weeks (60% content of high-concentrate to 40% forage; HC group), whereas the others received a low-concentrate diet ad libitum (40% high-concentrate content to 60% forage; LC group). Sustained low ruminal pH values (pH 5.6 maintained for 4 h/d) and reduced milk yield performance (2.07 kg/d less than LC cows) verified that SARA had been induced in the HC group. Results showed a significantly decreased concentrations of cis-9 monounsaturated long-chain fatty acids in plasma collected from hepatic but not portal veins. This was matched by reduced SCD1 mRNA and protein concentrations in HC livers. The expression levels of genes related to lipid formation (DGAT1 and PLIN2) were downregulated during SARA, whereas those of catabolic genes (CPT1A, CPT2, and ACOX1) and some inflammatory genes were upregulated. Expression of SCD1 was downregulated through reduced transcription and abundance of the transcription factor sterol regulatory element-binding protein 1 (SREBP1c).This effect was augmented by local chromatin tightening and DNA methylation at and around the SREBP1c binding site in the SCD1 promoter. Chromatin immunoprecipitation assays confirmed that SARA reduced SREBP1c binding at the SCD1 promoter; hence, epigenetic mechanisms are involved in regulating the expression of genes related to long-chain fatty acid modification, partially through downregulation of both SCD1 and SREBP1c in the liver. Our results suggest that in addition to inflammatory genes, SCD1 is also involved in SARA-induced epigenetic regulation and its associated metabolic changes. This knowledge might help to provide a target for intervening against the detrimental metabolic effects of SARA., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In the mammalian genome, approximately 50% of all genes are controlled by promoters with high GC contents. Analyzing the epigenetic mechanisms regulating their expression is difficult. Hence, we examined a method for stable quantification of such GC-rich DNA sequences. Quantification of DNA during real-time PCR is often based on reagent kits containing the fluorescent dye SYBR Green. However, these ready-made kits may not be suitable for amplifying DNA samples with a high GC content (>70%). DNA segments with eccentric GC contents are frequently found in proximal promoter areas, and their quantification may be necessary in chromatin accessibility by real-time polymerase chain reaction or chromatin immunoprecipitation analyses of epigenetic mechanisms of gene regulation. We therefore optimized the SYBR Green I FastStart reaction system by supplementing the system with dimethyl sulfoxide, betaine, and increased DNA polymerase content. Here, we describe the development of the assay and demonstrate its effectiveness for two different DNA templates, showing that these modifications allow for the reliable amplification and quantification of DNA with GC contents exceeding >70% using the LightCycler instrument.

Feeding rumen-protected fat (RPF) can improve energy supply for dairy cows but it affects glucose metabolism. Glucose availability is a precondition for high milk production in dairy cows. Therefore, this study investigated endocrine regulation of glucose homeostasis and hepatic gene expression related to glucose production because of RPF feeding in lactating cows. Eighteen Holstein dairy cows during second lactation were fed either a diet containing RPF (mainly C16:0 and C18:1; FD; n = 9) or a control diet based on corn starch (SD; n = 9) for 4 wk starting at 98 d in milk (DIM). Feed intake and milk yield were measured daily and milk composition once a week. Blood samples were taken weekly for analyses of plasma triglyceride, nonesterified fatty acids (NEFA), β-hydroxybutyrate, bilirubin, urea, lactate, glucose, insulin, and glucagon. At 124 DIM, an intravenous glucose tolerance test (GTT; 1g/kg of BW(0.75)) was performed after a 12-h period without food. Blood samples were taken before and 7, 14, 21, and 28 min after glucose administration, and plasma concentrations of glucose, insulin, and glucagon were measured. Glucose half-life as well as areas under the concentration curve for glucose, insulin, and glucagon were calculated. After slaughter at d 28 of treatment, liver samples were taken to measure mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose 6-phosphatase (G6Pase), and facilitative glucose transporter 2. Dry matter intake, but not energy and protein intake, was lower in FD than in SD. Milk yield during lactation decreased more in SD than in FD, and milk protein was lower in FD than in SD. Plasma concentrations of triglycerides and NEFA were higher in FD than in SD. Plasma insulin concentrations were lower and the glucagon:insulin ratios were higher in FD than in SD. Fasting glucose concentration before GTT was lower, and fasting glucagon concentrations tended to be higher in FD than in SD. In liver, fat content tended to be higher and G6Pase mRNA abundance was lower in FD than in SD. Lower hepatic G6Pase mRNA abundance was associated with reduced fasting plasma glucose concentrations, but the glucose-induced insulin response was not affected by RPF feeding. Hepatic G6Pase gene expression might be affected by DMI and might be involved in the regulation of glucose homeostasis in dairy cows, resulting in a lower hepatic glucose output after RPF feeding., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Coordination of the primary defense mechanisms against pathogens relies on the appropriate expression of pathogen recognition receptors (PRRs) triggering the early release of effector molecules of the innate immune system. To analyze the impact of this system on the counteraction of infections of the mammary gland (mastitis), we characterized the bovine gene encoding the key PRR Toll-like receptor 9 (TLR9) and mapped its precise position on chromosome BTA22. The sequence information was used to establish real-time PCR quantification assays to measure the mRNA abundances of TLR9, TLR2, and TLR4 together with those of beta-defensin 5 (BNBD5), an early bactericidal effector molecule of the innate system, in healthy and infected mammary glands. Mastitis strongly increased (4- to 13-fold) the mRNA abundances of all of these genes except TLR9. Slight subclinical infections already caused a substantial increase in the copy numbers, though they did so the least for TLR9. Induction was not systemic, since mRNA abundance was low in uninfected control quarters of the udder but high in the severely infected quarters of the same animal. The number of TLR2 copies correlated well with those of TLR4, indicating coordinated regulation of these two PRRs during infection of the udder. Their coordinated regulation explains our unexpected observation that pure Staphylococcus aureus infections caused a strong increase also in TLR4 mRNA abundance. In situ hybridizations revealed that BNBD5 is expressed predominantly in the mammary epithelial cells (MEC) of the infected gland. Our data therefore suggest a significant contribution of the innate immune system to counteract mastitis and attribute a prominent effector function to the MEC.

Unlabelled: Few studies have been done on the circadian rhythm of QT- und corrected QT-interval in diabetics., Method: 13 type II diabetics (mean duration 10.08 yrs +/- 1.95 (SEM), 12 insulin-dependent) and 13 hypertensives, mean age 58.2 +/- 197 (SEM) and 57.3 +/- 1.4 yrs (p = n.s.), respectively, without cardiovascular events within the last 6 months before rehabilitation underwent Holter monitoring. We investigated mean and maximum QT-interval, QTc-interval (Bazett-formula), RR-interval as well as spectral parameters LF, HF, and LF/HF ratio., Results: Both groups showed significant circadian variations of mean and maximum QT-intervals. No group differences were found. There was no significant circadian rhythm of mean or maximum QTc-interval. Diabetics had significantly prolonged maximum QTc-intervals during the day-time. Mean and maximum RR-intervals in diabetics were significantly shorter. Both groups showed a significant circadian rhythm. LF and HF were significantly shorter in diabetics. Only in diabetics could significant circadian rhythm of LF and HF be demonstrated. The LF/HF ratio was significantly shorter in diabetics. The hypertensive group showed a decreasing circadian trend whereas diabetics showed increasing LF/HF ratio trend., Conclusion: Mean and maximum QT-intervals are significantly shorter in diabetics than in hypertensives. Maximum QTc-interval prolongation is highly significant (p < 0.01) in diabetics. Spectral parameters LF and HF are reduced in comparison with hypertensives. The prolongation of the maximum QTc-interval could be expression of an increased arrythmogenic and cardiovascular risk in diabetics.

Activity of acetyl-CoA carboxylase (ACC)-alpha is rate limiting for de novo synthesis of fatty acids. The encoding gene is expressed by three different promoters. We characterized promoter III (PIII) from cow, previously only known from sheep. Quantitation of transcripts by RNAse protection assays and real time PCR revealed that PIII is primarily expressed and strongly induced ( approximately 28-fold) in the lactating mammary gland. PIII transcripts are expressed in mammary epithelial cells (MEC) as shown by in situ hybridization. A 2999 bp segment of the PIII promoter conferred prolactin and dexamethasone inducibility to a luciferase reporter gene in stably transfected mouse MEC cells. Lactogenic induction was abolished if a unique signal transducer and activator of transcription (STAT)-binding site at position -797 was inactivated by two point mutations. An oligonucleotide probe harboring this STAT-site specifically bound nuclear proteins from the lactating mammary gland. Binding was abolished by those two point mutations and super-shift analyses showed that STAT5A factors are present in this complex. Hence, prolactin, acting through STAT5, contributes to the activation of ACC expression in the milk producing cells of the lactating mammary gland. We discuss that STAT5 might be important in determining the milk composition by coordinating fatty acid and protein synthesis during lactation.

The enzyme acetyl-CoA carboxylase alpha (ACC-alpha) is rate-limiting for the synthesis of long-chain fatty acids de novo. As a first characterization of the bovine gene encoding this enzyme, we established the entire bovine ACC-alpha cDNA sequence (7041 bp) and used experiments with 5' rapid amplification of cDNA ends to determine the heterogeneous composition of 5' untranslated regions, as expressed from three different promoters (PI, PII and PIII). The individual locations of these promoters have been defined within an area comprising 35 kbp on Bos taurus chromosome 19 ('BTA19'), together with the segmentation of the first 14 exons. Primer extension analyses reveal that the nutritionally regulated PI initiates transcription from at least four sites. PI transcripts are much more abundant in adipose and mammary-gland tissues than in liver or lung. A 2.6 kb promoter fragment drives the expression of reporter genes only weakly in different model cells, irrespective of stimulation with insulin or dexamethasone. Thus bovine PI is basically repressed, like its analogue from rat. Finely graded deletions of PI map two separate elements, which have to be present together in cis to repress bovine PI. The distal component resides within a well-preserved Art2 retroposon element. Thus sequence, structure and evolutionary origin of the main repressor of PI in bovines are entirely different from its functional counterpart in rat, which had been identified as a (CA)(28) microsatellite. We show that, in different mammalian species, unrelated genome segments of different origins have been recruited to express as functionally homologous PI the ancient and otherwise highly conserved ACC-alpha-encoding gene.

The STAT transcription factors form a family of signal transducers and activators of transcription. We sequenced the bovine STAT5B cDNA and both STAT5-encoding genes, STAT5A and STAT5B, representing the first complete description of any STAT5-encoding gene. DNA fiber FISH hybridization revealed that the genes reside only 40 kbp apart on BTA19. Both genes are segmented into 19 exons and all but two of the homologous exons are of equal size. The genes harbor a central block of nearly identical DNA sequence (97.5% sequence identity over 3373 bp), spanning from intron 5 to intron 9. Isolation and sequencing of the homologous segments from mouse revealed the same unusually high degree of intronic sequence conservation in these segments of the murine STAT5-encoding genes. However, the respective sequences are completely divergent between the two species. A comparison of the inter- and intragenic cDNA sequence preservation at nonsynonymous sites reveals that the DNA-binding domain is under the strongest selection pressure for both intergenic and factor-specific intragenic sequence preservation. The so-called "SH3" segment of the linker domain, in contrast, shows species-specific sequence identity in all but one amino acid residues in both factors, in cattle, human, and mouse. This indicates that the same species-specific selection pressure occurs on the linker domain from both factors, STAT5A and STAT5B. Thus, the comparison of evolutionary selection pressures resting on various domains suggests that the DNA-binding domain might contribute to differential DNA binding of STAT5A and STAT5B factors, while both might interact equally well with other cellular factors through a segment of the linker domain.

According to the guidelines and standards for a comprehensive phase II rehabilitation a new outpatient/part-time outpatient program was developed and started at an existing rehabilitation center in January 1997. All patients of the new program were included in a follow-up study and compared with patients of the same center, who met the inclusion and exclusion criteria for the outpatient mode, but wanted to perform their rehabilitation in the full-time residential mode. Both groups were examined before, at the end and 6 months after the program. Until December 1998 118 patients after an acute cardiac event such as myocardial infarction, PTCA or heart surgery were rehabilitated in the outpatient/part-time outpatient mode. The short- and medium-term results concerning somatic outcomes, e.g. the risk factor profile or the improvement of the maximum work capacity, were equal in both groups. Comparing the direct costs for a 4-week rehabilitation, the part-time outpatient program was 26%, the outpatient program even 52% cheaper than the standard full-time residential program. The new program is as effective as the residential rehabilitation, but it is cheaper. Because of the in- and exclusion criteria it is suitable for only a subgroup of cardiac patients, because of the demands and standards the new program it can be offered only in special rehabilitation centers.

The transcription factors STAT5 mediates prolactin signals in mammary epithelial cells. The cDNA of bovine STAT5A was cloned, sequenced and compared to other species. The encoded protein proves to be > 95% homologous to other mammals. We show that the STAT5A mRNA of the closely related ovine species contains an extended (by 130 nt) 5'-untranslated region, being encoded by an extra-exon, and accounts, possibly, for improved translation efficiency.

Lysozyme-encoding genes (Lys) constitute a gene-family in ruminants. While several of these genes are highly expressed in stomach (sLys), few other copies are weakly expressed in other tissues, notably in polymorphnuclear granulocytes and macrophages (mLys). Searching an understanding for these grossly different levels of expression, we isolated the bovine variant of the gene being expressed in granulocytes and characterized it by sequencing, together with its promoter. Spanning about 9 kb of genomic DNA, the gene is found to be segmented into four exons, in common with all other Lys, as known from vertebrates. Sequence homologies between all bovine sLys-variants exceeds 70% over much of the entire coding sequence and promoter region. This indicates (i) that bovine lysozymes expressed either in stomach or granulocyte originate from a common ancestral gene and (ii) also excludes the possibility that the observed weak expression of the mLys gene is due to major structural rearrangements within the promoter segment. However, primer extension analysis based on RNA isolated from kidney locates the transcription startpoint (tsp of that gene) 44 nt further upstream than observed in both, bovine stomach lysozyme RNA or any of the homologous genes in mice and man. The observed weak expression of this bovine mLys gene appears to be a consequence of both the presence of an extra ATG codon in the extended 5'-UTR, and a severe down mutation of the ancestral TATA-box which is only partially compensated for by the presence of another mutation further upstream resulting in a weak substitute promoter sequence.

The isolation and nucleotide sequence determination of the 5' flanking region of the mouse growth hormone (mGH)-encoding gene (mGH) is described. The mGH gene consists of five exons and four introns, as is observed in other mammalian species. The second intron in mGH is much smaller than its rat counterpart, thus being similar in size to human, bovine and porcine GH. The transcription start point was determined to be a C residue 62 bp upstream from the start codon, ATG. Analysis of 1767 bp of the 5' flanking region, with respect to putative regulatory elements, revealed a TATA box, two binding sites for growth hormone factor (GHF1), a GC box (SP1), a thyroid-response element (TRE) and a silencer (SiL) sequence motif. As expected, the mGH promoter shows a higher degree of homology with rat, as compared to the other mammalian species like pig, cattle and human, where an overall homology exists only at the proximal promoter region.

cDNA copies of a bovine lysozyme (bLys)-encoding gene (Lys) were isolated from libraries specific for granulocytes, as well as the lactating mammary gland. Analysis of each of the longest Lys-specific cDNA inserts revealed nucleotide sequence identity over the entire overlap of 1418 bp. Incomplete at the 5' end, the combined sequence codes for 11 of the 18-amino-acid (aa) Lys leader peptide and 130 aa residues of the mature Lys. Similar to mouse and human Lys from blood cells, the encoded protein contains one aa residue more (Pro103) than any of the bLys derived from stomach. Furthermore, unlike any of the known bLys genes, our sequence reveals the copy of a bovine retroposon element in the 3' untranslated region (UTR) of the mRNA approximately at the same position where an Alu-retroposon element resides within the human copy of the gene. As a further distinction from bLys expressed in stomach, we identified a segment within the 3'UTR of the mRNA which is conserved between the bovine and human blood cell variants of the Lys, but does not have significant sequence homology to any of the bovine lysozyme genes known so far. By sequence comparisons, we present evidence that this segment has been deleted during evolutionary divergence of the stomach Lys. Hence, we describe the sequence of a heretofore unknown bLys, being expressed in granulocytes. Bearings of our observations on the understanding of Lys evolution are discussed, as well as the possibility that the product of this gene may be responsible for the functional Lys activity in bovine milk.