Inheritance of UMP synthase in dairy cattle

The inheritance of uridine-5'-monophosphate (UMP) synthase in dairy cattle was consistent with a two-allele, single-autosomal-locus model. Two phenotypes were associated with different levels of the enzyme in bovine erythrocytes. The predominant phenotype (assumed normal) had twice the concentration of UMP synthase as the second phenotype (deficient). A one-to-one correspondence between enzyme level and genotype identified one homozygote as normal, the heterozygote as deficient, and the other homozygote as unobserved. Three alternative hypotheses were rejected. The deficiency as homozygous recessive was rejected because 20 matings between assumed normal males and deficient females resulted in 10 normal and 10 deficient offspring. The hypothesis that the deficiency was homozygous dominant was rejected because the 95 percent confidence interval about the observed gene frequency, 0.0024 to 0.0146, did not contain the estimated gene frequency for equilibrium between an average 10(-5) mutation rate and selection against the deficiency as homozygous dominant. Analyses of female relatives implicated one bull as deficient (96 percent probability), as he had, independently, 2 deficient daughters, 5 deficient granddaughters from untested dams, and 3 deficient great-granddaughters from untested ancestors. The hypothesis that the deficiency was sex-linked was rejected because 3 of 9 tested sons of the putative deficient bull were deficient. Calf mortality is expected in 25 percent of matings between deficient animals.     

Developmental Genetics of the 2e-F Region of the Drosophila X Chromosome: A Region Rich in "Developmentally Important" Genes

We have analyzed the 2E1-3A1 area of the X chromosome with special attention to loci related to embryogenesis. Published maps indicate that this chromosomal segment contains ten bands. Our genetic analysis has identified 11 complementation groups: one recessive visible (prune), two female steriles and eight lethals. One of the female sterile loci is fs(1)k10 for which homozygous females produce both egg chambers and embryos with a dorsalized morphology. The second female sterile is the paternally rescuable fs(1)pecanex in which unrescued embryos have a hypertrophic nervous system. Of the eight lethal complementation groups two are recessive embryonic lethals: hemizygous giant (gt) embryos possess segmental defects, and hemizygous crooked neck (crn) embryos exhibit a twisted phenotype. Analysis of these mutations in the female germ line indicates that gt does not show a maternal effect, whereas normal activity of crn is required for germ cell viability. Analysis of the maternal effect in germ line clones of the remaining six recessive lethal complementation groups indicates that four are required for germ cell viability and one produces ambiguous results for survival of the germ cells. The remaining, l(1)pole hole, is a recessive early pupal lethal in which embryos derived from germ line clones and lacking wild-type gene activity exhibit the "torso" or "pole hole" phenotype.     

A mammalian spot test: induction of genetic alterations in pigment cells of mouse embryos with x-rays and chemical mutagens.

Embroys heterozygous for five recessive coat-color genes from the cross C 57 BL/6 J Han x T-stock were x-irradiated with 100/r o r treated in utero with 50 mg/3 kg methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), respectively. Controls consisted of irradiated embryos of C 57 BL x C 57 BL matings homozygous wild-type for the genes under study, and non-treated offspring of both types of mating. The colors of the spots were observed in the adult fur were either due to expression of the recessive coat genes or were white. I. Irradiated and mutagen-treated offspring of C 57 BL x T-stock matings had almost exclusively nonwhite spots, distributed randomly over the mouse surface. 2. Irraidated offspring of C 57 BL x C 57 BL matings had only white spots which were always midventral. 3. In non-treated offspring of both types of mating no spot could be observed. After correcting for white midventral spots observed in the one type of control, the frequency of expression of one or the other of the recessive color genes is calculated to be about 11% for embryos irradiated with 100r or 101/2 days postconception, about 1% for embryos irradiated with 100r at 9 days postconception, about &% for embryos treated with 50 mg MMS/kg at 101/2 days postconception, and about 8% for embryos treated with 50 mg EMS/2 days postconception. It is discussed that the white midventral spots are preferentially the result of pigment cell killing, while the nonwhite spots are preferentially the result of gene mutations or recombinational processes like mitotic crossing over and mitotic gene conversion. Of numerical and structural chromosome aberrations only those come into question which are able to pass the filter of several mitoses. Therefore, the test system described is supposted to cover not only heitable DNA-alterations, but the whole spectrum of them.     

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase

The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.     

Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.     

Description of an embryonic lethal gene, l(5)-1, linked to Wsh

A recessive lethal mutant linked to Wsh causes the death of homozygous embryos between 4.5 and 5.5 days postcoitum (pc). Histological examination of implantation sites from intercross and backcross matings indicates that homozygotes are not all evident at 4.5 days pc, when embryos have begun to form trophectoderm giant cells and primitive endoderm, but are degenerating by 5.5 days pc, with only a few primary giant cells remaining after this time. The mutants thus form blastocysts that initiate the implantation process but the inner cell mass and polar trophectoderm fail to develop further. In vitro examination and culture of blastocysts indicated that the mutant homozygotes hatch from the zona pellucida and outgrow, although they do so somewhat more slowly than normal embryos. After 3 days of culture, the inner cell masses of mutant outgrowths may be smaller than normal. Since the gene has no known heterozygous effect and the primary gene function remains unknown, the mutant has been given the provisional symbol l(5)-1 for the first lethal on chromosome 5.     

Embryonic loss in superovulated cattle caused by the 1;29 Robertsonian translocation

The effect of the 1;29 Robertsonian translocation on fertility was studied using embryos resulting from matings of nine carrier cows and two carrier bulls. Embryos were collected from the following three mating groups utilizing superovulation: normal bull cross normal cow, normal bull cross translocation carrier cow, and translocation carrier bull cross normal cow. The proportion of ova which were fertilized did not vary among the groups, indicating that fertilization rates were not affected by the translocation. The translocation cows did yield fewer embryos on average than did cows with normal karyotypes, which may suggest ovulation rates are reduced (at least after superovulation attempts) in cattle carrying the 1;29 translocation. Twenty of 39 embryos successfully karyotyped had abnormal chromosome complements. All four of the theoretically predicted karyotypes and two additional abnormal combinations were found. Eight of 39 (20.5%) embryos karyotyped had unbalanced karyotypes which would have resulted in embryonic loss. The proportion of embryos with unbalanced karyotypes, was slightly higher when the cow (36%) carried the translocation than when the bull (19%) did. Results of this study indicate that fertility is impaired due to the presence of this translocation. The major loss in reproductive potential appears to be due to embryonic loss rather than fertilization failure.     

Characterization of the female-sterile mutant Almondex of Drosophila melanogaster

Drosophila melanogaster females homozygous for the sex-linked recessive mutation almondex (amx) are completely sterile when mated to amx males. Matings of amx X+ yield low numbers of heterozygous female offspring, which frequently show abnormalities of the thorax and abdominal sternites, and an occasional non-disjunctional, non-mutant (XO) male offspring. The results of mating experiments reported here can be explained by assuming that the cytoplasm of eggs produced by amx females is deficient for some material that is necessary for normal development.Homozygous amx females have apparently normal reporductive organs and a high egg yield. Eggs are usually fertilized. In matings to amx males, all zygotes die as embryos; in matings to non-amx males, all ordinary (XY) male zygotes and most female zygotes die as embryos. Survival to the adult stage is more frequent at higher temperatures and, surprisingly, increases also with maternal age.     

Detection and prevalence of UMP synthase deficiency among dairy cattle

A partial deficiency of UMP synthase has been detected in Holstein dairy cattle. Since affected females secrete milk with elevated concentrations of orotate, milk orotate was used to screen for the condition among 880 cows in 17 randomly selected Holstein herds in Illinois. Mean orotate was 85.0 +/- 1.5 microgram/ml milk and 17 cows had milk orotate in excess of 170 micrograms/ml. Including the latter animals, 42 cows were evaluated for erythrocyte UMP synthase and 15 were found to be partially deficient. Thus, at least 1.7 percent of all cows had the condition; this is a minimal estimate because the initial screen was milk orotate and this may be low, particularly early in lactation. Deficient cows had half the level of UMP synthase as normal, nondeficient cows (1.30 +/- 0.06 vs. 2.79 +/- 0.10 units/ml). The binomial classification of deficient versus normal accounted for 72 percent of the variation noted in UMP synthase. Milk orotate was significantly elevated in deficient cows (337.8 +/- 31.3 micrograms/ml), validating its use as a screening device. Urinary orotate also was higher for deficient cows (28.4 +/- 5.2 vs. 9.2 +/- 0.8 micrograms/ml) and differentiated the two groups as well as milk orotate. Normal and deficient cows did not differ in milk lactose concentrations. Erythrocyte UMP synthase also was measured in 85 Holstein bulls used for artificial insemination and 6 had low levels of UMP synthase (1.39 +/- 0.19 vs. 2.92 +/- 0.05 units/ml); the binomial classification accounted for 42 percent of the variation. The partially deficient animals identified appear to be heterozygotes for a condition expected to be lethal in the homozygous state.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Impact of asynchronous ovulations on the expression of sex-dependent growth rate in bovine preimplantation embryos

In cattle, male embryos have a faster growth rate than female embryos, and this results in alteration of the normal 1:1 sex ratio in embryos divided into three developmental groups. The fastest developed one-third are predominantly males, the slowest one-third predominantly females, and in the intermediate one-third no alteration of the sex ratio is seen, However, the deviations of the sex ratios are only 15-20% from random. These findings are compatible with the assumption that, in superovulated cows, ovulations follow a normal distribution and that, at the time of sampling at Day 7, male and female embryos differ with regard to development by 1 or 2 h. Because of this it is unlikely that larger changes in the sex ratios can be expected.     

Characterization of UMP synthase in dairy cattle heterozygous for a lethal recessive trait

UMP synthase was characterized biochemically in dairy cattle heterozygous for a deficiency of this enzyme. Both activities comprising this bifunctional enzyme are decreased, with OMP decarboxylase more affected than orotate phosphoribosyltransferase. Immunotitration of UMP synthase activity revealed the presence of the protein product of the mutant allele in the heterozygous animals. UMP synthases from normal and deficient cattle were not distinguished from one another by kinetic constants, responses to inhibitors, pH profiles, or thermal lability. It was concluded that the 50% reduction in enzyme activity in heterozygous cattle is the result of the presence of only half the normal level of catalytically active UMP synthase.     

Bovine protoporphyria: documentation of autosomal recessive inheritance and comparison with the human disease through measurement of heme synthase activity.

Protoporphyria is an autosomal dominant disease in man in which protoporphyrin accumulated because of a defect in heme synthase (ferrochelatase) activity. A disease has been described in cattle that has the same manifestations as does the human disease. We measured heme synthase activity in sonicates of cultured skin fibroblasts and whole liver homogenates from animals with protoporphyria, their unaffected parents, and normal cattle in order to examine the mode of inheritance and compare it with human protoporphyria. The mean activity (+/- SEM) in fibroblasts from the three groups was 2.0 +/- 0.4, 47 +/- 12, and 149 +/- 10 pmol heme formed/mg protein per hr, respectively, consistent with autosomal recessive inheritance. Similarly, the levels of heme synthase activity in livers of the parents were intermediate to those of normal animals and of animals with protoporphyria. When compared with normal human fibroblasts and liver, the specific activity of heme synthase in normal bovine tissue was significantly higher. These studies indicate that manifestations of protoporphyria do not occur in cattle unless the animal is homozygous for the gene defect, whereas in humans, the heterozygous condition is sufficient. This is probably because the specific activity of heme synthase in cells of heterozygous animals is not reduced to a level that significantly alters heme metabolism.     

Developmental Genetics of the 2c-D Region of the Drosophila X Chromosome

We have conducted a genetic and developmental analysis of genes within the 2C-D area of the X chromosome. Phenotypes of 33 mutations representing nine adjacent complementation groups including eight recessive lethals and one visible homeotic mutation (polyhomeotic) are described. Germline clonal analysis of the eight zygotic lethals has revealed three types of gene requirements: normal activity at two pupal lethal loci (corkscrew and C204) and one larval lethal locus (ultraspiracle) is required for normal embryogenesis; normal activity at three larval lethal loci (DF967, VE651 and Pgd) is required for normal oogenesis; and activity at only one locus (EA82), a larval lethal, appears to have no maternal requirement. Ambiguous results were obtained for the GF316 lethal complementation group. Analysis of mitotic figures of the pupal lethals indicates that C204 disrupts an essential mitotic function. This result correlates with the preblastoderm arrest observed among embryos derived from germline clones of C204. Embryos derived from germline clones of corkscrew (csw) exhibit a "twisted" phenotype. The recessive lethal ultraspiracle (usp) disrupts the organization of the posterior tip of the larval both zygotically and maternally: second instar usp/Y larvae derived from heterozygous usp/+ mothers possess an extra set of spiracles, whereas usp/Y embryos derived from females possessing a germline clone (usp/usp) exhibit a localized ventral defect in the ninth or posterior eighth abdominal segment. Analysis of the phenotypes of deficiency-hemizygous embryos indicates the presence of an embryonic zygotic lethal locus, as yet unidentified, which produces central nervous system and ventral hypoderm degeneration. Additional information on the genetic organization of loci within the adjacent 2E area are also described.(ABSTRACT TRUNCATED AT 250 WORDS)     

The extended survival of tw5/tw5 mouse embryo cells in vitro

The tw5 haplotype is a recessive mutation which is lethal when homozygous in mouse embryos following implantation. This series of studies was undertaken to determine the effect of the tw5/tw5 genotype on embryos developing in vitro. Blastocyst embryos from +/tw5 inter se matings were compared with control blastocysts obtained from matings between T/+ and +/+ females and +/tw5 males for their abilities to continue development in vitro in two culture media. The data show that there are no significant differences between the percentages of experimental and control blastocyst embryos which attach and outgrow or which contain inner cell masses on any day of culture up to equivalent gestation day 21 in either media. These findings show that the life span of cells from tw5/tw5 embryos can be extended significantly by in vitro culture.     

Nanomelic chondrocytes synthesize a glycoprotein related to chondroitin sulfate proteoglycan core protein

Chicken embryos homozygous for the autosomal recessive gene nanomelia exhibit cartilage defects, synthesize low levels of cartilage chondroitin sulfate proteoglycan (CSPG), and are missing the CSPG core protein (Argraves, W. S., McKeown-Longo, P. J., and Goetinck, P. F. (1981) FEBS Lett. 131, 265). In our studies of nanomelic chondrocytes in culture, we detected neither sulfate-labeled CSPG nor its Mr 370,000 core protein. However, in immunoprecipitation reactions using both polyclonal and monoclonal antibodies directed against the cartilage CSPG core protein, we identified a protein of Mr 300,000 that contains an epitope found in the hyaluronic acid-binding region of the normal core protein. This protein was also detected among products synthesized by chondrocytes obtained from phenotypically normal embryos resulting from matings between parents heterozygous for nanomelia. Sensitivity to endoglycosidase H indicated that the product is a glycoprotein with attached mannose-rich oligosaccharides. Pulse-chase studies revealed the disappearance of the glycoprotein after 6 h of chase, but no detectable formation of proteoglycan. Our results suggest that although nanomelic chondrocytes are deficient in the production of normal CSPG and its core protein, they do synthesize a smaller, immunologically related glycoprotein that does not undergo the post-translational processing characteristic of the normal cartilage core protein.     

Expression of a new mutation (Axd) causing axial defects in mice correlates with maternal phenotype and age

A new autosomal mutation, Axd (axial defects), is described. Axd segregates in a simple Mendelian fashion, and it is dominant with incomplete penetrance and variable expressivity. The phenotype of Axd heterozygotes ranges from a variety of tail anomalies to visibly normal tails. Approximately 12% of neonates from curly-tail (CT) F1 (Axd/+) x F1 (Axd/+) matings exhibit open neural tube defects (NTD) in the lumbosacral region and 16% have curly tails. Mean litter sizes and resorption rates comparable to wild type indicate that homozygosity for Axd is not obligately lethal. Genetic background plays a major role in Axd expression. Strains such as BALB/cByJ allow the highest penetrance of the mutation in single dose (46%), whereas, in CF-1 mice Axd is recessive. The tail phenotype of heterozygous Axd/+ dams, in part reflective of their genetic background, correlates with the incidence of NTD in F2 offspring: CT mothers produce significantly more neonates with frank NTD than normal tail mothers. At the one embryonic period examined for this study (D13/D14 post-coitus), an 85% higher incidence of total axial defects is observed than among the F2 at birth. Unchanging litter size and the relative increase in phenotypically normal offspring by birth suggest that Axd acts by delaying posterior neural tube closure. One of the most significant findings in this study is that maternal age influences the survival of Axd embryos in utero. Axd/+ dams older than 8 months yield fewer mean implants, higher resorption rates, and fewer viable embryos with axial defects than do Axd/+ dams younger than 8 months. Axd is not allelic to nor linked to the Sp (splotch) gene which also affects neurulation.     

Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families.

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a lambdaEMBL-3 human genomic library and report a single-copy gene spanning approximately 15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5' flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A-to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A (v = .26) and 440Gpoly (v = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function.     

Cranial effects of retinoic acid in the loop-tail (Lp) mutant mouse

The effects of retinoic acid (RA) on the manifestation and nature of neural tube defects (NTD) in heterozygous embryos of mutant mice carrying the gene loop-tail (Lp) and in normal (+/+) littermates and embryos from normal homozygous matings were compared with NTD that occur in untreated abnormal homozygous (Lp/Lp) embryos. A single intraperitoneal dose (5 mg/kg) of RA administered at 9 AM or 3 PM on day 8 of gestation induced NTD in +/+ as well as Lp/+ embryos removed on day 12 of gestation. All of the NTD were confined to the brain and consisted of exencephaly involving the diencephalon, mesencephalon, and metencephalon. In neither phenotype (Lp/+; +/+) was the massive exencephaly and myeloschisis characteristic of untreated Lp/Lp embryos produced; thus, it is possible that the teratogenic mechanisms of RA-induced defects and of Lp-induced defects may differ.