Genome-Wide DNA Methylation and Its Effect on Gene Expression During Subclinical Mastitis in Water Buffalo.

Subclinical mastitis (SCM) in buffalo is one of the most challenging paradoxes for the dairy sector with very significant milk production losses and poses an imminent danger to milch animal's milk-producing ability. We present here the genome-wide methylation specific to SCM in water buffalo and its consequential effect on the gene expression landscape for the first time. Whole-genome DNA methylation profiles from peripheral blood lymphocytes and gene expression profiles from milk somatic cells of healthy and SCM cases were catalogued from the MeDIP-Seq and RNA-Seq data. The average methylation in healthy buffaloes was found to be higher than that in the SCM-infected buffaloes. DNA methylation was abundant in the intergenic region followed by the intronic region in both healthy control and SCM groups. A total of 3,950 differentially methylated regions (DMRs) were identified and annotated to 370 differentially methylated genes (DMGs), most of which were enriched in the promoter region. Several important pathways were activated due to hypomethylation and belonged to the Staphylococcus aureus infection, Th17 cell differentiation, and antigen processing and presentation pathways along with others of defense responses. DNA methylome was compared with transcriptome to understand the regulatory role of DNA methylation on gene expression specific to SCM in buffaloes. A total of 4,778 significant differentially expressed genes (DEGs) were extracted in response to SCM, out of which 67 DMGs were also found to be differentially expressed, suggesting that during SCM, DNA methylation could be one of the epigenetic regulatory mechanisms of gene expression. Genes like CSF2RB, LOC102408349, C3 and PZP like, and CPAMD8 were found to be downregulated in our study, which are known to be involved in the immune response to SCM. Association of DNA methylation with transposable elements, miRNAs, and lncRNAs was also studied. The present study reports a buffalo SCM web resource (BSCM2TDb) available at http://webtom.cabgrid.res.in/BSCM2TDb that catalogues all the mastitis-related information of the analyses results of this study in a single place. This will be of immense use to buffalo researchers to understand the host-pathogen interaction involving SCM, which is required in endeavors of mastitis control and management.

Retiform hemangioendothelioma: an uncommon pediatric vascular neoplasm.

Retiform hemangioendothelioma is considered as a low grade angiosarcoma, commonly seen in 2(nd)-4(th) decade of life. The youngest patient reported is of 9 years of age. A 9-year-old boy was presented with an asymptomatic red patch on the chest since 1 year. There was an erythematous patch with nodule of about 5 mm near the distal margin. Biopsy from the nodule revealed numerous thin-walled branching vessels in dermis resembling rete testis, suggestive of retiform hemangioendothelioma. We, hereby, report this case for its rarity and uncommon clinical presentation in childhood period.

Insight into evolution of a giant congenital nevomelanocytic nevus over 14 years.

Giant congenital nevomelanocytic nevus (GCNN) is a rare variant of congenital melanocytic nevus measuring >20 cm in size that often has a garment-like distribution. Regular follow up is recommended because of a risk of melanoma transformation of 4.6%. We report a 14-year-old boy with gradual regression of giant congenital melanocytic nevus over the left upper limb, chest, back and axilla, whom we have followed-up since birth. At birth, a hyperpigmented jet-black patch without hair was present over the left side of torso and upper limb including palms and nails. Follow up at the ages of 1, 5, 11 and 14 years showed progressive spontaneous regression of the nevus resulting in shiny atrophic skin, diffuse hypopigmentation, lentigo-like macules, nodules and arthrogryphosis of affected areas. Histopathology of the lesions on follow-up revealed absence of pigmented nevus cells in the regressing areas and thickened sclerotic collagen bundles.

A novel modification of the autoimplantation therapy for the treatment of multiple, recurrent and palmoplantar warts.

BACKGROUND: Ideal treatment for warts should be effective, safe, have less morbidity and provide long-lasting immunity against human papilloma virus. This can optimally achieved by the stimulation of the immune system against the virus. The autoimplantation of warts, autowart injection and quadrivalent vaccines have been used for this purpose. Autoimplanatation is a simple technique where the subcutis deep wart tissue is harvested as a donor and implanted into the uninvolved skin. However, this led to two wounds, at donor and recipient sites. AIM: The aim was to evaluate the safety and efficacy of a novel modification of autoimplantation therapy in the treatment of multiple, recurrent and palmoplantar warts. SUBJECTS AND METHODS: Thirty-three patients with multiple, recurrent and palmoplantar warts were enrolled. Instead of taking a bit of the wart tissue, the donor tissue was harvested by paring the wart. The pared tissue was implanted deep into the subcutis by stab incision done using the same surgical blade no. 11. The resolution of all warts within 3 months after the procedure was considered successful. Patients with complete clearance were followed up for 1 month for any recurrence. RESULTS: Out of 35 patients, 27 patients were available for follow-up. A total of 20 (74.1%) patients showed a complete clearance of warts within 3 months. Partial clearance was seen in 1 patient. Erythematous nodules developed at the site of implantation in 3 (11.1%) patients. There was relapse in one patient. CONCLUSION: A modified technique of autoimplantation of warts employing the pared stratum corneum tissue from the wart is a simple, effective, less traumatic and rapid procedure in the treatment of multiple, recurrent and palmoplantar warts.

SAG/ROC/Rbx/Hrt, a zinc RING finger gene family: molecular cloning, biochemical properties, and biological functions.

The RING (really interesting new gene) finger proteins containing a characteristic C3HC4 or C3H2C3 motif appear to act as E3 ubiquitin ligase and play important roles in many processes, including cell-cycle progression, oncogenesis, signal transduction, and development. This review is focused on SAG/ROC/Rbx/Hrt (sensitive to apoptosis gene/regulator of cullins/RING box protein), an evolutionarily conserved RING finger family of proteins that were cloned recently by several independent laboratories through differential display, yeast two-hybrid screening, or biochemical purification. SAG/ROC2/Rbx2/Hrt2 is expressed in multiple mouse adult tissues, as well as early embryos. In humans, both SAG and ROC1 are ubiquitously expressed at a very high level in heart, skeletal muscle, and testis. Expression of both SAG and ROC1 is induced by mitogenic stimulation. SAG is also induced by a redox agent in cultured cells, as well as in in vivo mouse brain upon ischemia/reperfusion. Structurally, SAG consists of four exons and three introns with at least one splicing variant and two pseudogenes. The SAG gene promoter is enriched with multiple transcription factor binding sites. Biochemically, SAG binds to RNA, has metal-ion binding/free radical scavenging activity, and is redox-sensitive. Most importantly, like ROC1, SAG/ROC2 binds to cullins and acts as an essential component of E3 ubiquitin ligase. Biologically, SAG is a growth-essential gene in yeast. In mammalian cells, SAG protects apoptosis mainly through inhibition of cytochrome c release/caspase activation, and promotes growth under serum deprivation at least in part by inhibiting p27 accumulation. Blocking SAG expression via antisense transfection inhibits tumor cell growth. Thus, SAG appears to be a valid drug target for anticancer therapy.

SAG/ROC2/Rbx2/Hrt2, a component of SCF E3 ubiquitin ligase: genomic structure, a splicing variant, and two family pseudogenes.

We have recently cloned and characterized an evolutionarily conserved gene, Sensitive to Apoptosis Gene (SAG), which encodes a redox-sensitive antioxidant protein that protects cells from apoptosis induced by redox agents. The SAG protein was later found to be the second family member of ROC/Rbx/Hrt, a component of the Skp1-cullin-F box protein (SCF) E3 ubiquitin ligase, being required for yeast growth and capable of promoting cell growth during serum starvation. Here, we report the genomic structure of the SAG gene that consists of four exons and three introns. We also report the characterization of a SAG splicing variant (SAG-v), that contains an additional exon (exon 2; 264 bp) not present in wildtype SAG. The inclusion of exon 2 disrupts the SAG ORF and gives rise to a protein of 108 amino acids that contains the first 59 amino acids identical to SAG and a 49-amino acid novel sequence at the C terminus. The entire RING-finger domain of SAG was not translated because of several inframe stop codons within the exon 2. The SAG-v protein was expressed in multiple human tissues as well as cell lines, but at a much lower level than wildtype SAG. Unlike SAG, SAG-v was not able to rescue yeast cells from lethality in a ySAG knockout, nor did it bind to cullin-1 or have ligase activity, probably because of the lack of the RING-finger domain. Finally, we report the identification of two SAG family pseudogenes, SAGP1 and SAGP2, that share 36% or 47% sequence identity with ROC1/Rbx1/Hrt1 and 30% or 88% with SAG, respectively. Both genes are intronless with two inframe stop codons.

Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation.

The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation.

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins. Oncogene (2000) 19, 2855 - 2866

Expression, purification, and biochemical characterization of SAG, a ring finger redox-sensitive protein.

We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis. Here we report the expression, purification, and biochemical characterization of SAG. Bacterially expressed SAG is brown in color and dithiothreitol (DTT)-sensitive. SAG forms large oligomers without DTT that can be reduced into a monomer in the presence of DTT. These features help us to purify SAG using the chromatography with or without DTT. Likewise, purified SAG is redox sensitive. Upon H2O2 exposure, SAG forms oligomers as well as monomer doublets due to the formation of the inter- or intramolecular disulfide bonds, respectively. This process can be reversed by DTT or prevented by pretreatment with the alkylating reagent, N-ethylmaleimide (NEM). Although SAG contains two putative heme-binding sites and a RING finger domain, the protein appears not to bind with heme and to lack transcription factor activity as determined in a Gal4-fusion/transactivation assay. Wildtype, but not RING finger domain-disrupted SAG mutants, prevents copper-induced lipid peroxidation. These results, along with our previous observations, suggest that SAG is an intracellular antioxidant molecule that may act as a redox sensor to buffer oxidative-stress induced damage.

Transcriptional activation of the human glutathione peroxidase promoter by p53.

Glutathione peroxidase (GPX) is a primary antioxidant enzyme that scavenges hydrogen peroxide or organic hydroperoxides. We have recently found that GPX is induced by etoposide, a topoisomerase II inhibitor and a p53 activator. In a search for a cis-element that confers potential p53 regulation of GPX, we identified a p53 binding site in the promoter of the GPX gene. This site bound to purified p53 as well as p53 in nuclear extract activated by etoposide. A luciferase reporter driven by a 262-base pair GPX promoter fragment was transcriptionally activated by wild type p53 in a p53 binding site-dependent manner. The same reporter was also activated in a p53 binding site-independent manner by several p53 mutants. The p53 binding and transactivation of the GPX promoter were enhanced by etoposide in p53-positive U2-OS cells. Etoposide-induced transactivation was blocked by a dominant negative p53 mutant, indicating that endogenous wild type p53, upon activation by etoposide, transactivated the GPX promoter. Furthermore, expression of endogenous GPX was induced significantly at both mRNA and enzyme activity levels by etoposide in U2-OS cells but not in p53-negative Saos-2 cells. This is the first report demonstrating that GPX is a novel p53 target gene. The finding links the p53 tumor suppressor to an antioxidant enzyme and will facilitate study of the p53 signaling pathway and antioxidant enzyme regulation.

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents.

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.

Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.

Coagulation factor VIII (FVIII) and factor V are homologous glycoproteins that have a domain structure of A1-A2-B-A3-C1-C2. FVIII is a heterodimer of the heavy chain (domains A1-A2-B) and the light chain (domains A3-C1-C2) in a metal ion-dependent association between the A1- and A3-domains. Previous studies identified a 110-amino acid region within the FVIII A1-domain that inhibits its secretion and contains multiple short peptide sequences that have potential to bind immunoglobulin-binding protein (BiP). FVIII secretion requires high levels of intracellular ATP, consistent with an ATP-dependent release from BiP. Site-directed mutagenesis was used to elucidate the importance of the potential BiP-binding sites in FVIII secretion. Mutation of Phe at position 309 to Ser or Ala enhanced the secretion of functional FVIII and reduced its ATP dependence. The F309S FVIII had a specific activity, thrombin activation profile, and heat inactivation properties similar to those of wild-type FVIII. However, F309S FVIII displayed increased sensitivity to EDTA-mediated inactivation that is known to occur through metal ion chelation-induced dissociation of the heavy and light chains of FVIII. The results support that Phe309 is important in high affinity heavy and light chain interaction, and this correlates with a high affinity BiP-binding site. Introduction of the F309S mutation into other secretion defective FVIII mutants rescued their secretion, demonstrating the ability of the this mutation to improve secretion of mutant FVIII proteins retained in the cell.

Biosynthesis, assembly and secretion of coagulation factor VIII.

Factor VIII is a large complex glycoprotein that is deficient in hemophilia A. It has a domain organization consisting of A1-A2-B-A3-C1-C2 where the B domain is a heavily glycosylated region that is dispensable for procoagulant activity. Factor VIII expression is 10-to 20-fold lower than the homologous coagulation factor V. Factor VIII expression is limited due to a low level of steady-state messenger RNA in the cytoplasm and inefficient transport of the primary translation product from the endoplasmic reticulum to the Golgi apparatus. Within the secretory pathway, factor VIII is processed to a heterodimer of the heavy chain (domains A1-A2-B) in a metal ion association with the light chain (domains A3-C1-C2). Upon secretion from the cell, von Willebrand factor binds the light chain of factor VIII and stabilizes the factor, preventing degradation. Protein folding within the mammalian secretory pathway is facilitated by molecular chaperones. Within the endoplasmic reticulum, factor VIII exhibits stable interaction with protein chaperones identified as the immunoglobulin-binding protein (BiP), calnexin and calreticulin. BiP is a peptide-dependent ATPase that interacts with exposed hydrophobic surfaces on unfolded proteins or unassembled protein subunits. A potential BiP binding site within factor VIII has been identified. Mutation of a single amino acid residue in the potential BiP binding site increased the secretion efficiency of factor VIII by threefold. Interestingly, the proposed BiP binding site is adjacent to a type-1 copper binding site within the A1 domain that is required for interaction between the factor VIII A1 domain and the A3 domain. We propose that Cu(I) binds the type-1 copper ion-binding site in the A1 domain and provides the essential requirement for a stable interaction between the heavy and light chains. Calnexin and calreticulin are transmembrane and lumenal proteins, respectively, localized to the endoplasmic reticulum, which associate transiently with many soluble and membrane glycoproteins during folding and subunit assembly. The calnexin and calreticulin interaction with factor VIII occurs primarily through amino-terminal linked oligosaccharides within the heavily glycosylated factor VIII B domain and this interaction appears to be required for factor VIII secretion. The findings suggest that factor VIII cycles through interactions with BiP, calnexin and calreticulin. Although the interaction with BiP does not appear to be required for factor VIII secretion, data suggest that the calnexin and/or calreticulin interaction is required for secretion. The observations suggest a unique requirement for carbohydrate processing and calnexin/calreticulin interaction that may limit the productive secretion of factor VIII and have implications for approaches towards somatic cell gene therapy for hemophilia A.

Depletion of manganese within the secretory pathway inhibits O-linked glycosylation in mammalian cells.

Proteins transiting the secretory pathway are posttranslationally modified by addition of oligosaccharides to asparagine N-linked and serine and threonine O-linked residues. The effects of divalent cation depletion on oligosaccharide processing of erythropoietin (EPO) and macrophage colony stimulating factor (M-CSF) were studied in Chinese hamster ovary cells. Treatment with A23187 did not inhibit M-CSF or EPO secretion but did inhibit addition of complex N-linked and O-linked oligosaccharides to both molecules. Similar results were obtained by treatment with thapsigargin, a potent inhibitor of the Ca(2+)-activated microsomal ATPase, indicating that the effect was due to depletion of divalent cations within the secretory pathway. Whereas addition of extracellular calcium chloride did not reverse the inhibition in complex N-linked and O-linked glycosylation, addition of manganese chloride partially reversed both defects. These results are consistent with a specific manganese requirement within the secretory pathway for the processing of complex N-linked oligosaccharides and the addition of O-linked oligosaccharides. Since there are no known specific inhibitors of O-linked glycosylation, the use of ionophores should significantly facilitate studies on the requirement and role of O-linked oligosaccharides in protein structure and function.

Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues.

We have used RNA in situ hybridization to study the regional expression of the Huntington's disease gene (HD) and its rat homologue in brain and selected nonneural tissues. The HD transcript was expressed throughout the brain in both rat and human, especially in the neurons of the dentate gyrus and pyramidal neurons of the hippocampal formation, cerebellar granule cell layer, cerebellar Purkinje cells and pontine nuclei. Other brain areas expressed lower levels of the HD transcript without pronounced regional differences. Neuronal expression predominated over glial expression in all regions. HD mRNA was also expressed in colon, liver, pancreas and testes. The regional specificity of neuropathology in HD, which is most prominent in the basal ganglia, thus cannot be accounted for by the pattern of expression of HD.

Human cystathionine beta-synthase cDNA: sequence, alternative splicing and expression in cultured cells.

Cystathionine beta-synthase (CBS) deficiency is the major cause of homocystinuria in humans. The most frequent symptoms of homocystinuria include: dislocated optic lenses, vascular disorders, skeletal abnormalities and mental retardation. Patients with this deficiency have elevated levels of homocyst(e)ine, methionine and low cysteine in their body fluids. These abnormal levels often partially or fully normalize upon treatment with pharmacological doses of vitamin B6. To investigate the molecular and biochemical basis for these conditions, it was necessary to determine the nucleotide and polypeptide sequence of CBS. We report here the human CBS cDNA sequence of 2,554 nucleotides encoding the CBS subunit of 551 amino acids. An intron of 214 bp appears to be retained in the 3'-untranslated region of most of the fibroblast and liver mRNA. We also report a frequent Mspl polymorphism in the 3'-untranslated sequence and two synonymous mutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A). The amino acid sequence similarity of human and rat CBS is greater than 90%; the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyase from bacteria and plants. Lastly, we demonstrate that expression of the human enzyme in CHO cells yields enzymatically active protein of the expected size with a half-life of approximately 14 hrs.

An alternatively-spliced mRNA in the carboxy terminus of the neurofibromatosis type 1 (NF1) gene is expressed in muscle.

The gene for neurofibromatosis type 1 (NF1) was identified by positional cloning and found to contain two alternatively spliced exons. The first described alternatively spliced exon (exon 23a) is located within the GAP-related domain of the gene and inserts an additional 63 nucleotides into the NF1 mRNA. The second alternatively spliced exon (exon 48a) is located near the extreme carboxy terminus of the gene and inserts an additional 54 nucleotides into the mRNA. This second isoform, termed 3'ALT, was originally detected while screening a fetal brain cDNA library. Examination of its expression by reverse-transcribed RNA PCR demonstrates high level of expression in cardiac muscle, skeletal muscle and smooth muscle. Trace levels of expression are detected in brain and nerve. The 3'ALT isoform is expressed in fetal cardiac muscle, adult left ventricle and cardiac Purkinje cells. Further confirmation of the existence of this isoform was obtained by blotting the PCR products with a radiolabeled oligonucleotide entirely derived from sequences contained within exon 48a and by direct sequencing of the PCR products. Additionally, this isoform is expressed in muscle tissues from other vertebrate species. The expression of this isoform in muscle suggests that the NF1 gene may play additional tissue-specific roles in muscle development and signal transduction.

Magnetic bead capture of expressed sequences encoded within large genomic segments.

Magnetic bead capture utilizes biotin-streptavidin magnetic bead technology to isolate cDNAs rapidly from large genomic intervals, giving several thousand-fold enrichment of the selected cDNAs. The technique can allow parallel analysis of several large genomic segments of varying complexities and can be applied to the isolation of expressed sequences from various tissue sources.

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.