Propionicacidemia: absence of alpha-chain mRNA in fibroblasts from patients of the pccA complementation group.

Propionicacidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl-CoA carboxylase (PCC) activity. The enzyme has the structure alpha 4 beta 4, with the alpha chain containing a covalently bound biotin prosthetic group. Patients have been placed into two major complementation groups, pccA and pccBC, that may correspond to the genes encoding the alpha and beta chains of PCC. The pccBC group is further divided into two subgroups, pccB and pccC, apparently owing to intragenic complementation. We previously reported combined alpha- and beta-chain deficiency in pccA mutants and absence of beta chain in pccC and pccBC mutants after isotope-tracer labeling and immunoprecipitation of cultured-fibroblast extracts. Using cDNA clones coding for the alpha and beta chains as probes, we found absence of alpha mRNA in four of six pccA strains and presence of beta mRNA in all pccA mutants studied. We also found presence of both alpha and beta mRNAs in three pccBC, two pccB, and three pccC mutants. From these data, we confirm the gene assignments of the complementation groups (PCCA gene = pccA complementation group; PCCB gene = pccBC and subgroups) and support the view that pccA patients synthesize a normal beta chain that is rapidly degraded in the absence of complexing with alpha chains.     

Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups.

Propionicacidemia is a metabolic disorder resulting from a deficiency of propionyl-CoA carboxylase activity. The enzyme is composed of two polypeptides: a 72,000-dalton alpha chain which contains the biotin ligand and a 56,000-dalton beta chain. It has been suggested that the two major complementation groups in this disorder, pccA and pccBC (with subgroups pccB and pccC), correspond to the genes encoding these two chains. To correlate gene product with complementation groups, 15 mutant and four normal human fibroblast strains were analyzed by [35S]methionine and [3H]biotin labeling. Immunoprecipitation and gel electrophoresis of the polypeptides revealed that alpha chains are synthesized by mutants of pccBC and both subgroups but not in four out of five pccA mutants. On the other hand, beta chains were detected only in pccB mutants. We suggest that pccA encodes the alpha chain of PCC while pccBC encodes the beta chain, and furthermore predict that the beta chain is unstable in the absence of the alpha chain.     

Variability in the amount of beta-globin mRNA in beta0 thalassemia

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.     

Cloning of functional alpha propionyl CoA carboxylase and correction of enzyme deficiency in pccA fibroblasts.

Propionyl CoA carboxylase (PPC) is a heteromeric enzyme composed of alpha subunits (PCCA) and beta (PCCB) subunits. We describe cDNA clones expressing human PCCA and complementation of the genetic defect in pccA fibroblasts by DNA-mediated gene transfer. Two cDNA clones were constructed. The first corresponds to the previously reported, putatively full-length, open reading frame. The second encodes a chimera composed of the mitochondrial leader sequence of human methylmalonyl CoA mutase and the mature PCCA protein. Both clones reconstitute propionate flux to normal levels in fibroblasts from patients genetically deficient in PCCA (pccA). The maximal level of propionate flux approached, but never exceeded, the levels seen in control plates of normal cells. In contrast, the maximal level of PPC holoenzyme activity reached only 10%-20% that of normal controls, which corresponded roughly to the fraction of cells actually transformed with the recombinant gene. These data suggest that the level of PCCA expression in fibroblasts does not normally limit PCC holoenzyme activity or propionate flux. The fact that a small fraction of cells reconstitutes propionate flux to normal levels suggests that metabolic cooperation between cells is capable of increasing the metabolic capacity of recombinant enzyme in a subpopulation of cells. These factors may have important implications for the rational design of somatic gene therapy for PCCA deficiency.     

Imbalanced synthesis of human choriogonadotropin alpha and beta subunits reflects the steady state levels of the corresponding mRNAs

Previous studies have demonstrated an imbalance in placental levels of the human choriogonadotropin (hCG) alpha and beta subunits. Free alpha subunit was present in first trimester placentae, and the imbalance was accentuated as gestation approached parturition. Two sets of experiments were performed to assess the control on production levels of each subunit. Synthesis of the alpha and beta subunits was assessed by labeling the nascent chains of polysomes derived from first trimester placenta. The products of these reactions were immunoprecipitated with subunit-specific antisera and the labeled subunits were quantitated; the ratio of alpha to beta subunit synthesized was 1.7. To examine whether this imbalanced synthesis reflected differences in the amount of subunit mRNAs, or differing mRNA translational efficiencies, the ratio of the steady state levels of these mRNAs was also determined. Total first trimester placental RNA was hydrolyzed with alkali, 5'-end-labeled with 32P, and hybridized in DNA excess to cloned alpha and beta cDNAs. These experiments demonstrated the presence of twice as much hCG-alpha mRNA as hCG-beta mRNA. In term placenta, the amounts of excess alpha subunit are greater than at first trimester; the ratio of alpha to beta mRNAs in term RNA was about 12:1. Thus, the subunit mRNA levels are independently regulated and their imbalance accounts for differences in the quantities of alpha and beta subunits seen in placental tissue.     

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.     

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.     

Expression of myosin heavy chains during thyroid hormone-induced cardiac growth

The expression of mRNAs for two cardiac myosins has been studied in the ventricles of hypo- and hyperthyroid rabbits by using cloned cDNA sequences corresponding to the mRNAs of the alpha- and beta-myosin heavy chains (HCs). The temporal change in relative levels of the alpha and beta HC mRNAs after triiodothyronine (T3) treatment of hypothyroid rabbits was determined by nuclease S1 mapping. In the hypothyroid state, only NC beta-mRNA was expressed in the ventricles. The HC alpha-mRNA was first detectable 4 h after administration of T3 (200 micrograms/kg) to hypothyroid animals. By 12, 24, and 72 h, HC alpha-mRNA represented 20, 50, and 90% of total myosin mRNA. The relationship between the relative mRNA levels and relative synthesis rates of myosin HCs was evaluated in 5- to 6-wk-old normal and thyrotoxic rabbits. Myosin synthesis rates were determined by labeling of protein in vivo with [2H]leucine. The V1 (HC alpha) and V3 (HC beta) isomyosins were separated by immune affinity chromatography and the HCs were isolated electrophoretically. In a normal euthyroid group of animals and in animals 12 and 24 h after administration of 200 micrograms of thyroxine, the relative mRNA levels and relative synthesis rates of the alpha and beta HCs were not significantly different. Our results show that, first, thyroid hormone causes a rapid accumulation of HC alpha-mRNA and loss of HC alpha-mRNA, and second, in normal and thyrotoxic rabbits, the relative synthesis rates of HC alpha and HC beta reflect the relative abundance of their respective mRNAs. These data are consistent with the thyroid hormones regulating synthesis of ventricular myosin at steps that precede translation of its message.     

Hexosaminidase isozyme in type O Gm2 gangliosidosis (Sandhoff-Jatzkewitz disease).

The residual enzyme of the fibroblasts of a child with homozygous type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) has been found to correspond with a minor fraction of enzyme which can be isolated from normal fibroblasts by repeated chromatography. This enzyme is designated as hexosaminidase (hex) S. It reacts with antiserum prepared against homogeneous hex A but not with serum prepared against homogeneous hex B. These findings support our previously described model of the relationship between hex A and hex G: hex A has the structure (alpha beta)3, while hex B is (beta)6. Type B GM2 gangliosidosis (Tay-Sachs disease) is the alpha- mutation, while type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) is the beta- mutation. In the absence of normal beta subunits there is increased polymerization of alpha subunits forming hex S, which probably has a structure of (alpha)6. A parallel between the thalassemias and GM2 gangliosidosis is evident: deficiency of one of the chains of which the protein is composed leads to an excess of polymers comprised of the other chains. In type B GM2 gangliosidosis, the excess of beta chanis leads to increased amounts of hex B beta)6; in type 0 GM2 gangliosidosis, the excess of alpha chains leads to formation of increased amounts of the alpha chain polymer, hex S.     

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta, and not alpha, subunit mRNA

In this paper we establish the response of LLC-PK1/Cl4 cells, a pig kidney cell line, to incubation in medium containing 0.25 mM K+. The amounts of the Na,K-ATPase alpha and beta subunits, determined by Western blot, increase coordinately to greater than 2-fold over control by 24 h in low K+ and remained elevated for the duration of the study period (48 h). Na,K-ATPase activity, measured enzymatically, increased 1.4-fold by 24 h and remained elevated. In order to determine if this response was initiated pretranslationally, alpha and beta subunit mRNA levels were determined by Northern blot analysis. While there was no change in alpha-mRNA levels, beta levels increased significantly, to 1.9-fold over control by 6 h of treatment and remained elevated. This selective increase in beta-mRNA was accompanied by 1.6- and 3.1-fold increases in the respective rates of accumulation of newly synthesized alpha and beta subunits, assessed by immunoprecipitating subunits from pulse-labeled cells. The degradation rates of mature Na,K-ATPase subunits did not change during 16 h of exposure to low K+, but after 16 h there was a selective decrease in the alpha degradation rate, relative to control. These results suggest that increased pretranslational regulation of the beta subunit alone is sufficient to increase accumulation of both alpha and beta subunits. These findings support the notion that in LLC-PK1 cells newly synthesized beta is rate-limiting and thus regulates, through alpha beta assembly, the number of pumps transported to the plasma membrane.     

Globin mRNA translation on Artemia salina ribosomes with components from Friend leukemia cells.

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cytst of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCl ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin. The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the alpha and beta chains of globin. The alpha:beta chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4:1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1:4, thus these two fractions appear to provide sources enriched in either alpha or beta globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethysulfoxide-stimulated Friend leukemia cells when translated in vitro produced alpha and beta chains in a ratio of 1:1. These peptides are formed in the same ratio in the intact cells.     

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus.

The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI alpha and LHI beta, encoded by the pufA (LHI alpha) and pufB (LHI beta) genes. Pulse-labeling experiments showed that in the presence of the LHI alpha polypeptide, the LHI beta polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI alpha polypeptide. Each of the pufA and pufB genes was deleted to test whether the LHI alpha and beta polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI beta polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI alpha polypeptide was present. However, the LHI beta polypeptide did not accumulate in the membrane in the absence of the LHI alpha protein but was degraded linearly within about 12 min. In contrast to the LHI beta protein, only trace amounts of the LHI alpha polypeptide were inserted into or attached to the membrane if the LHI beta polypeptide was not synthesized.     

Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.     

Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase

Fatty acid synthesis in pea chloroplasts is regulated by light/dark. The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation. Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT). To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state. Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides. Similar results were confirmed in the full-length CT. These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits. Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo. A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light.     

Mutations participating in interallelic complementation in propionic acidemia.

Deficiency of propionyl-CoA carboxylase (PCC; alpha 4 beta 4) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA (alpha-subunit) and PCCB (beta-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, we have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound heterozygote of Pro228Leu and Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS + 1 G-->T, located after Lys466. We suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, we previously had proposed that delta Ile408 was the complementing allele. We now show that its second allele, "Ins.Del," a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. We conclude that the interallelic complementation results from mutations in domains that can interact between beta-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, we suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site.     

抗、感绿豆象绿豆种子差异蛋白的分离与鉴定

为寻找抗绿豆象绿豆种子中的重要抗虫成分,以抗虫绿豆晋绿7号、B20及感虫绿豆潍绿2117为试验材料,采用蛋白质双向电泳(2-DE)和质谱技术,对抗、感虫绿豆中差异蛋白及功能进行了鉴定与分析。结果表明,抗、感虫绿豆中差异蛋白表达量超过2.5倍的点共有15个。其中6个蛋白点通过数据库得到了成功鉴定,涉及3种蛋白质,分别为8S球蛋白(α亚型和β亚型)、核酮糖1,5-二磷酸羧化酶/加氧酶(RuBis CO)亚基结合蛋白和合成淀粉酶与胰蛋白酶两种抑制剂的前体多肽链。蛋白点B49(即8S球蛋白α亚型)和B31(RuBis CO亚基结合蛋白)在抗、感虫绿豆中的差异表达量分别达到10 000倍和23倍。抗虫绿豆中8S球蛋白α亚型及β亚型、RuBis CO伴侣蛋白及胰蛋白酶抑制剂的前体作为抗虫物质影响了绿豆象的生长发育甚至导致其死亡,与抗虫性的量效关系及联合效应还需进一步验证。     

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.