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PubMed2026

Activity of natural single nucleotide variants of human alkyladenine DNA glycosylase AAG R145H, G163S and R197C involved in DNA binding.

Alkyladenine DNA glycosylase (AAG) is a critical enzyme in the base excision repair (BER) pathway that safeguards genome integrity by removing structurally diverse alkylated and deaminated purine lesions from DNA. It serves as a primary defense against alkylation-induced mutations, which are linked to cancer development, chronic inflammation, and neurodegenerative diseases. Single nucleotide variants (SNVs) in the gene coding region have the potential to alter the enzyme's functionality, potentially modulating the repair capacity and affecting response and prognosis following chemoradiotherapy. In our study, we investigated three SNVs that lead to amino acid class changes in regions involved in DNA substrate coordination: R145H, G163S, and R197C using an in vitro approach. Using biochemical assays and molecular dynamics simulations, we evaluated the thermal stability, DNA binding affinity, and glycosylase activity of AAG variants toward hypoxanthine (Hx) and 1,N6-ethenoadenosine (εA) containing substrates. The G163S variant showed reduced thermal stability due to the conformational strain in the β-hairpin loop that intercalates in DNA, but retained εA excision activity comparable to that of wild-type AAG, while losing activity against Hx-containing DNA. The R197C variant had a four-fold reduction in DNA binding affinity for both substrates, and was catalytically inactive, unable to excise either damaged bases. This loss of function correlated with the rearrangement of the 201-210 loop and the reorientation of Arg-201 and Arg-207, which disrupts critical DNA contacts. However, the R145H variant retained near-wild-type thermal stability and activity on both substrates, despite bioinformatic predictions of deleterious effect. Molecular dynamics simulations revealed variant-specific structural disruptions. The data obtained underscore the importance of experimental validation in assessing the functional impact SNVs.

DNA Glycosylases↗
PubMed1994

Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini.

1. The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2. Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3' untranslated sequence. 3. The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4. Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-1 protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5. PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.

Adenylyl Cyclases↗
PubMed1992

Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2).

This study was designed to identify the target molecules of the natural killer (NK) cell-mediated recognition of normal allogeneic target cells. As previously shown, the gene(s) governing the first NK-defined allospecificity (specificity 1) were found to be localized in the major histocompatibility complex region between BF gene and HLA-A. In addition, the analysis of a previously described family revealed that a donor (donor 81) was heterozygous for three distinct NK-defined allospecificities (specificities 1, 2, and 5). HLA variants were derived from the B-Epstein-Barr virus cell line of donor 81 by gamma irradiation followed by negative selection using monoclonal antibodies specific for the appropriate HLA allele. Several variants were derived that lacked one or more class I antigen expressions. These variants were analyzed for the susceptibility to lysis by NK clones recognizing different allospecificities. The loss of HLA-A did not modify the phenotype (i.e., "resistance to lysis"). On the other hand, a variant lacking expression of all class I antigens became susceptible to lysis by all alloreactive clones. Variants characterized by the selective loss of class I antigens coded for by the maternal chromosome became susceptible to lysis by anti-2-specific clones. Conversely, variants selectively lacking class I antigens coded for by paternal chromosome became susceptible to lysis by anti-1 and anti-5 clones (but not by anti-2 clones). Since the Cw3 allele was lost in the variant that acquired susceptibility to lysis by anti-2 clones and, in informative families, it was found to cosegregate with the character "resistance to lysis" by anti-2 clones, we analyzed whether Cw3 could represent the element conferring selective resistance to lysis by anti-2 clones. To this end, murine P815 cells transfected with HLA Cw3 (or with other HLA class I genes) were used as target cells in a cytolytic assay in which effector cells were represented by alloreactive NK clones directed against different specificities. Anti-2-specific clones efficiently lysed untransfected or A2-, A3-, and A24-transfected P815 cells, while they failed to lyse Cw3-transfected cells. NK clones recognizing specificities other than specificity 2 lysed untransfected or Cw3-transfected cells. Thus, the loss of Cw3 resulted in the de novo appearance of susceptibility to lysis, and transfection of the HLA-negative P815 cells with Cw3 resulted in resistance to lysis by anti-2 clones. Therefore, we can infer that Cw3 expression on (both human and murine) target cells confers selective protection from lysis mediated by anti-2 NK clones.

Alleles↗
PubMed2000

Apolipoprotein E polymorphisms and concentration in chronic diseases and drug responses.

Apolipoprotein (apo) E is an important circulating and tissue protein involved in cholesterol homeostasis and many other functions. The common polymorphism in the coding region of the gene, four polymorphisms in the promoter region, other additional single nucleotide polymorphisms, as well as several apo E variants have been identified. The common coding polymorphism strongly influences the lipid metabolism and the circulating concentration of apo E itself. This polymorphism is at the origin of the implication of apo E in cardiovascular and neurodegenerative diseases, but also of the relation of apo E with longevity. Probably due to its many metabolic and functional consequences, apo E polymorphism has been shown to influence the responses of patients to several drugs (fibrates, statins, hormone replacement therapy, anti-Alzheimer drugs) or environmental interventions (black tea, alcohol, diet). Apo E genotyping may be clinically helpful in defining the risk of patients and their responses to therapeutics. Finally, circulating apo E concentration appears to be altered in diseases and can be modulated by some of the drugs cited above. This parameter can thus also give interesting clinical information and could be a therapeutic target, providing it is validated. At the present time, we cannot exclude that apo E concentration may be the most prominent apo E parameter to be considered in health and disease, while apo E polymorphisms would represent only secondary parameters influencing apo E concentration.

Alleles↗
PubMed1989

Production of plasmids giving high expression of recombinant DNA-derived ovine growth hormone variants in Escherichia coli.

A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E. coli is described. The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3' to the initiation codon. Clones producing GH (with varying N-terminal extensions) were identified by immunological screening. Levels of expression of GH-related protein, measured by immunoassay or on SDS-polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein. The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.

Amino Acid Sequence↗
PubMed1999

Comparison of variant-specific hybridization and single-strand conformational polymorphism methods for detection of mixed human papillomavirus type 16 variant infections.

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.

Base Sequence↗
PubMed2005

Candidate gene association studies of genes involved in neuronal cholinergic transmission in Alzheimer's disease suggests choline acetyltransferase as a candidate deserving further study.

Consistent deficits in the cholinergic system are evident in the brains of Alzheimer's Disease (AD) patients, including reductions in the activities of acetylcholine, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), increased butyrylcholinesterase (BChE) activity, and a selective loss of nicotinic acetylcholine receptors (nAChRs). Accordingly, we have analyzed polymorphisms in the genes encoding AChE, ChAT, BChE, and several of the subunit genes from neuronal nAChRs, for genetic associations with late-onset AD. A significant association for disease was detected for a non-coding polymorphism in ChAT (allele chi(1) (2) = 12.84, P = 0.0003; genotype chi(2) (2) = 11.89, P = 0.0026). Although replication analysis did not confirm the significance of this finding when the replication samples were considered alone (allele chi(1) (2) = 1.02, P = 0.32; genotype chi(2) (2) = 1.101, P = 0.58) the trends were in the correct direction and a significant association remained when the two sample sets were pooled (allele chi(1) (2) = 12.37, P = 0.0004; genotype chi(2) (2) = 11.61, P = 0.003). Previous studies have reported significant disease associations for both the K-variant of BChE and the coding ChAT rs3810950 polymorphism with AD. Replication analyses of these two loci failed to detect any significant association for disease in our case-control samples.

Aged↗
PubMed1996

Preoperative imaging of lower extremity varicose veins: color coded duplex sonography or venography.

We prospectively examined 137 limbs in 112 consecutive patients with clinical evidence of severe varicosis by color coded duplex sonography and ascending venography (including varicography in 48 limbs) to evaluate the diagnostic capabilities of color coded duplex sonography in the assessment of venous anatomy, variant varicosis, postthrombotic changes, and incompetence of the superficial and perforating venous system. Additionally, descending venography was performed in the first 52 limbs and compared to color coded duplex sonography in the diagnosis of deep and superficial venous reflux. Variant venous anatomy (21 cases) was missed in two limbs and misinterpreted in one limb by ascending venography compared to surgery. Color coded duplex sonography was inconclusive in two cases. Variant varicosis (59 cases) was missed in seven surgically proved cases by venography and in one case by color coded duplex sonography. Color coded duplex sonography was inconclusive in five cases. Ascending venography was slightly superior to color coded duplex sonography in the detection of postphlebitic changes. Good agreement was found between color coded duplex sonography and descending venography in the grading of superficial (k = 0.75) and deep venous reflux (k = 0.79). Excellent agreement was found between ascending venography in the grading of long (k = 0.96) and short (k = 0.94) saphenous vein reflux. More incompetent perforating veins were detected by ascending venography, (and varicography) than by color coded duplex sonography, but the latter technique allows direct preoperative marking of the skin, which is beneficial for the surgeon. We conclude that color coded duplex sonography is a valuable imaging tool before venous stripping and is capable of replacing invasive ascending and descending venography. Only patients with inconclusive color coded duplex sonographic results (e.g., complex variant venous anatomy) should proceed to venography.

Adult↗
PubMed1990

Isolation and functional expression of pituitary peptidylglycine alpha-amidating enzyme mRNA. A variant lacking the transmembrane domain.

We demonstrate that, in rat pituitary, peptidylglycine alpha-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.

Amino Acid Sequence↗
PubMed2025

Novel genetic determinants contribute to hearing loss in a central European cohort with enlarged vestibular aqueduct.

BACKGROUND: The enlarged vestibular aqueduct (EVA) is the most commonly detected inner ear malformation. Biallelic pathogenic variants in the SLC26A4 gene, coding for the anion exchanger pendrin, are frequently involved in determining Pendred syndrome and nonsyndromic autosomal recessive hearing loss DFNB4 in EVA patients. In Caucasian cohorts, the genetic determinants of EVA remain unknown in approximately 50% of cases. We have recruited a cohort of 32 Austrian patients with hearing loss and EVA to define the prevalence and type of pathogenic sequence alterations in SLC26A4 and discover novel EVA-associated genes. METHODS: Sanger sequencing, single nucleotide polymorphism (SNP) assays, copy number variation (CNV) testing, and Exome Sequencing (ES) were employed for gene analysis. Cell-based functional and molecular assays were used to discriminate between gene variants with and without impact on protein function. RESULTS: SLC26A4 biallelic variants were detected in 5/32 patients (16%) and monoallelic variants in 5/32 patients (16%). The pathogenicity of the uncharacterized SLC26A4 protein variants was assigned or excluded based on their ion transport function and cellular abundance. The monoallelic or biallelic Caucasian EVA haplotype was detected in 7/32 (22%) patients, but its pathogenicity could not be confirmed. X-linked pathogenic variants in POU3F4 (2/32, 6%) and biallelic pathogenic variants in GJB2 (2/32, 6%) were also found. No CNV of SLC26A4 and STRC genes was detected. ES of eleven undiagnosed patients with bilateral EVA detected rare sequence variants in six EVA-unrelated genes (monoallelic variants in SCD5, REST, EDNRB, TJP2, TMC1, and two variants in CDH23) in five patients (5/11, 45%). Cell-based assays showed that the TJP2 variant leads to a mislocalized protein product forming dimers with the wild-type, supporting autosomal dominant pathogenicity. The genetic causes of hearing loss and EVA remained unidentified in (14/32) 44% of patients. CONCLUSIONS: The present investigation confirms the role of SLC26A4 in determining hearing loss with EVA, identifies novel genes in this pathophysiological context, highlights the importance of functional testing to exclude or assign pathogenicity of a given gene variant, proposes a possible diagnostic workflow, suggests a novel pathomechanism of disease for TJP2, and highlights voids of knowledge that deserve further investigation.

Humans↗
PubMed1999

Spatial codes and the hardness of string folding problems.

We present a general technique for proving NP-hardness (under randomized polynomial time reductions) of string folding problems over a finite alphabet. All previous such intractability results have required an unbounded alphabet size. These problems correspond to the protein folding problem in variants of the hydrophobic-hydrophilic (or HP) model with a fixed number of monomer types. Our proof also establishes the MAX SNP-hardness of these problems (again under randomized polynomial time reductions). This means that obtaining even an approximate solution to the protein folding problem, to within some fixed constant factor, is NP-hard. Our technique involves replacing the symbols of an unbounded alphabet by code-words over a fixed alphabet, and has two novel aspects. The first is the essential use of the approximation hardness of the source problem in the reduction, even for the proof of NP-hardness. The second is the concept of spatial codes, a variant of classical error-correcting codes in which different codewords are required to have large "distance" from one another even when they are arbitrarily embedded in three-dimensional space.

Computational Biology↗
PubMed1993

Detection of sequence variations in the human insulin-receptor gene by parallel denaturing gradient gel electrophoresis.

We developed a parallel denaturing gradient gel electrophoresis (DGGE) protocol to maximize the detection of nucleotide variants in the DNA sequence coding for the mature insulin receptor and in splice site junctions. The melting behaviours of exons 2 to 22 and flanking intronic sequences were computer-simulated using two programs, MELT87 and SQHTX. The data obtained from computer analysis were used to select primers for amplification by polymerase chain reaction and optimal electrophoretic conditions. The ability of this protocol to detect nucleotide changes at the insulin-receptor locus was assessed by studying amplified DNA of a patient with leprechaunism whose insulin-receptor mutations were known and by screening the insulin-receptor gene for polymorphisms in a population of unrelated caucasian individuals. Our results demonstrate that this DGGE protocol is sensitive since it detected (1) sequence variants reported to be undetectable by means of parallel DGGE, (2) previously characterized insulin-receptor nucleotide variants, and (3) unreported polymorphisms at the insulin-receptor locus of caucasian individuals. It is also simple as perpendicular denaturing gradient gels are not required. Application of this protocol will facilitate the search for molecular defects underlying the pathogenesis of insulin resistance observed in genetic syndromes of severe insulin resistance as well as in other metabolic disorders. In addition, its ability to detect several regions of the insulin-receptor gene displaying a number of common polymorphic sites and being multiallelic will contribute to linkage studies in families with diabetic and/or insulin-resistant subjects.

Base Sequence↗
PubMed2001

Sequence variation in the human uracil-DNA glycosylase (UNG) gene.

Spontaneous deamination of cytosine results in a premutagenic G:U mismatch that may result in a GC-->AT transition during replication. The human UNG-gene encodes the major uracil-DNA glycosylase (UDG or UNG) which releases uracil from DNA, thus, initiating base excision repair to restore the correct DNA sequence. Bacterial and yeast mutants lacking the homologous UDG exhibit elevated spontaneous mutation frequencies. Hence, mutations in the human UNG gene could presumably result in a mutator phenotype. We screened all seven exons including exon-intron boundaries, both promoters, and one intron of the UNG gene and identified considerable sequence variation in cell lines derived from normal fibroblasts and tumour tissue. None of the sequence variants was accompanied by significantly reduced UDG activity. In the UNG gene from 62 sources, we identified 12 different variant alleles, with allele frequencies ranging from 0.01 to 0.23. We identified one variant allele per 3.8kb in non-coding regions, but none in the coding region of the gene. In promoter B we identified four different variants. A substitution within an AP2 element was observed in tumour cell lines only and had an allele frequency of 0.10. Introduction of this substitution into chimaeric promoter-luciferase constructs affected transcription from the promoter. UDG-activity varied little in fibroblasts, but widely between tumour cell lines. This variation did not however correlate with the presence of any of the variant alleles. In conclusion, mutations affecting the function of human UNG gene are seemingly infrequent in human tumour cell lines.

Cells, Cultured↗
PubMed1991

Analysis of the glycoprotein gene of Tacaribe virus and neutralization-resistant variants.

We have previously generated neutralization-resistant variants of Tacaribe virus in the presence of a monoclonal antibody (MAb) specific for the envelope glycoprotein. The envelope glycoprotein precursor (GPC) genes of two variant viruses were sequenced following polymerase chain reaction amplification of a specific region of the Tacaribe virus S RNA, and compared with the GPC gene of the parental virus. Multiple nucleotide changes in the 3' half of the GPC gene were identified in the variants, suggesting that this part of the gene codes for the envelope glycoprotein of Tacaribe virus recognized by the MAb. Both variants showed unique amino acid substitutions up to 166 residues apart, suggesting that the most likely basis for neutralization resistance was a change in an epitope in which the critical residues are juxtaposed by conformation rather than by proximity in coding.

Amino Acid Sequence↗
PubMed2002

The BK channel beta1 subunit gene is associated with human baroreflex and blood pressure regulation.

BACKGROUND: The baroreflex, which is important for the minute-to-minute regulation of blood pressure and heart rate, is influenced by genetic variance. Ion channels are important to baroreflex afferent and efferent function. Mice missing the beta1 subunit of the Ca2+-sensitive potassium channel (BK) are hypertensive and have a reset baroreflex. We tested the hypothesis that variants in the gene (KCNMB1) coding for the BK beta1 subunit are associated with baroreflex function. METHODS: We studied six single-nucleotide polymorphisms (SNPs) in KCNMB1. RESULTS: Four SNPs in intron 3, exon 4a, exon 4b and exon 4c gave significant results. For instance, exon 4b SNP AA individuals had higher heart rate variability, compared to CA, or CC persons, in particular in the high-frequency range. The low-frequency range showed no association. Consistent with the heart rate variability data, homozygous AA persons had greater baroreflex slopes than CA or CC persons, also in the high-frequency range. These associations could not be shown in the low-frequency range for heart rate variability and baroreflex slopes. CONCLUSIONS: These data support the notion that variants in channel genes may be responsible for the great range in heart rate variability and baroreflex function observed in humans. Such variation may also play a role in the development of hypertension.

Adult↗
PubMed2000

Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients.

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.

Aged↗
PubMed1990

Characterisation of the alpha-1-antitrypsin M3 gene, a normal variant.

By sequence analysis of the complete protein-coding region of the human alpha-1-antitrypsin gene using polymerase chain reaction techniques, we have characterised one of the normal variants, M3. We have identified a single point mutation between M1 Val213 and M3 at codon position 376 which is a GAA(Glu) to GAC(Asp) transversion.

Amino Acid Sequence↗
PubMed1997

Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.

Amino Acid Sequence↗