Sam1 Gene Research Articles

Molecular evaluation of Cryptosporidium spp. among breeding calves of Lorestan province Western Iran.

Cryptosporidium spp. are opportunistic intestinal protozoans with global distribution and are of great importance as zoonotic protozoans are common to humans and domestic animals, including cattle and calves. Identification and detection of parasite species using precise methods including molecular methods can be an effective step in treating and controlling parasites. This study aimed to investigate the prevalence of Cryptosporidium among breeding calves of Khorramabad city, Lorestan province, Western Iran, using PCR. The faecal samples were taken from 181 healthy and diarrhoeal calves and after the Ziehl Neelsen Acid-fast staining and microscopic evaluation, the genomic DNA was extracted for molecular evaluations. To detect Cryptosporidium species, specific primers targeting the SAM-1 gene of Cryptosporidium and a commercial master mix were used for PCR. Out of 181 faecal samples of breeding calves in Khorramabad city, 9 samples (5%) were positive for Cryptosporidium spp. using the PCR method. Statistical analysis of the data showed that there was no significant statistical relationship between Cryptosporidium infection of the calves and variables of age, breed, type of water consumption, clinical signs of diarrhoea, and sampling location, while parasite infection had a significant relationship with calf gender so that all Cryptosporidium positive samples were from male calves (p ≤ 0.05). Considering the presence of Cryptosporidium infection, the region's traditional grazing system, and the close relationship between livestock and humans, there is a possibility of human infection in the region. So preventive measures such as periodic animal testing with sensitive and accurate diagnostic techniques including PCR, pharmacological treatment of livestock, water hygiene and the use of industrial grazing instead of traditional grazing to improve the hygiene of food consumed by livestock are recommended.

Interactions between SAM and the 5' UTR mRNA of the sam1 gene regulate translation in S. pombe.

The 5′ untranslated region (5′ UTR) of eukaryotic mRNA plays an important role in translation. Here we report the function of the 5′ UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe. Reporter assays, binding and chemical probing experiments, and mutational analysis show that the 5′ UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs plays an important role in eukaryotic gene regulation.

Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea.

Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers). Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously

Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.

Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.

A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR

A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps: (1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers), (3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis of the entire DNA of interest by PCR with outermost primers and template DNA from step 3, (5) post-extension (ten cycles of PCR at 72 degrees C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously.

Overexpression of a heterologous sam gene encoding S-adenosylmethionine synthetase in flax (Linum usitatissimum) cells: Consequences on methylation of lignin precursors and pectins.

The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens. This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells. The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins. Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells. Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls. The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.

Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA.

With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).

Different expression of an S‐adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis

Transformed callus cultures of Nicotiana tabacum were generated in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the tobacco callus cultures. Three distinct phenotypic classes were identified among the transgenic cell lines in relation to growth of the cells, structure of the calli, and level of SAM. Transgene silencing was observed in several cultivated transgenic calli and this phenomenon was correlated directly with a low level of SAM-1 mRNA accompanied by a decrease of the SAM-S activity. The transgenic calli overexpressing the SAM-1 gene accumulated a high SAM level. The modifications in SAM-S activity were reflected in the pattern of secondary products pres- ent in the different cell lines, thereby demonstrating that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 11–20, 2000.

Different expression of anS-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis

Transformed callus cultures of Nicotiana tabacum were generated in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the tobacco callus cultures. Three distinct phenotypic classes were identified among the transgenic cell lines in relation to growth of the cells, structure of the calli, and level of SAM. Transgene silencing was observed in several cultivated transgenic calli and this phenomenon was correlated directly with a low level of SAM-1 mRNA accompanied by a decrease of the SAM-S activity. The transgenic calli overexpressing the SAM-1 gene accumulated a high SAM level. The modifications in SAM-S activity were reflected in the pattern of secondary products present in the different cell lines, thereby demonstrating that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering.

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe.

By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the fission yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2-5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.

Polyamine Content and Somatic Embryogenesis in Papaver somniferum Cells Transformed with sam-1 Gene

Summary Papaver somniferum calluses transformed with the sam-1 gene from Arabidopsis thaliana, which encodes a SAM-synthetase, were subcultured over a 4-year period. The stability of the expression as well as the level of SAM synthetase activity were evaluated in 5 transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and in the control. All transgenic cell lines exhibited a level of SAM-synthetase activity higher than that of the control. The enhancement of the enzyme activity has been confirmed by Northern blot analyses. The level of polyamines (putrescine, spermidine, spermine and 1,3-diaminopropane) was also evaluated in cell lines cultured either in Linsmaier and Skoog medium containing growth regulators (LS) or on a hormone free medium (LSHF). Cell lines cultured in LS medium showed a variable yield of the polyamine content. Putrescine was the major polyamine. After transfer to a hormone free medium, all of the cell lines, except STSIV, were able to form embryo-like structures. In this condition, the polyamine level decreases about 4-fold, spermidine being the most abundant.

Cryopreservation does not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells

Transgenic cell lines of Papaver somniferum have been obtained via Agrobacterium tumefaciens. Papaver somniferum is known to be genetically unstable in in vitro culture conditions. Cryopreservation at ultra-low temperature is an appropriate strategy for long-term preservation of germplasm. We have studied the effects of osmotic stress due to cryoprotectants during pretreatment and of storage at –196 °C on the stability and the expression of the foreign sam1 gene from Arabidopsis thaliana. We established that the integrity, transcription of the transgene and enzymatic activity of its product were not affected by cryopreservation in liquid nitrogen

Potential retroviruses in plants: Tat1 is related to a group of Arabidopsis thaliana Ty3/gypsy retrotransposons that encode envelope-like proteins.

Tat1 was originally identified as an insertion near the Arabidopsis thaliana SAM1 gene. We provide evidence that Tat1 is a retrotransposon and that previously described insertions are solo long terminal repeats (LTRs) left behind after the deletion of coding regions of full-length elements. Three Tat1 insertions were characterized that have retrotransposon features, including a primer binding site complementary to an A. thaliana asparagine tRNA and an open reading frame (ORF) with approximately 44% amino acid sequence similarity to the gag protein of the Zea mays retrotransposon Zeon-1. Tat1 elements have large, polymorphic 3' noncoding regions that may contain transduced DNA sequences; a 477-base insertion in the 3' noncoding region of the Tat1-3 element contains part of a related retrotransposon and sequences similar to the nontranslated leader sequence of AT-P5C1, a gene for pyrroline-5-carboxylate reductase. Analysis of DNA sequences generated by the A. thaliana genome project identified 10 families of Ty3/gypsy retrotransposons, which share up to 51 and 62% amino-acid similarity to the ORFs of Tat1 and the A. thaliana Athila element, respectively. Phylogenetic analyses resolved the plant Ty3/gypsy elements into two lineages, one of which includes homologs of Tat1 and Athila. Four families of A. thaliana elements within the Tat/Athila lineage encode a conserved ORF after integrase at a position occupied by the envelope gene in retroviruses and in some insect Ty3/gypsy retrotransposons. Like retroviral envelope genes, this ORF encodes a transmembrane domain and, in some insertions, a putative secretory signal sequence. This suggests that Tat/Athila retrotransposons may produce enveloped virions and may be infectious.

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S‐adenosyl‐L‐methionine synthetase activity

We have developed a procedure for opium poppy (Papaver somniferum) transformation using Agrobacterium tumefaciens‐mediated gene delivery. Hypocotyl‐derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101(pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β‐glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the sam1 gene encoding the enzyme S‐adenosyl‐L‐methionine (SAM) synthetase and the nptII gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens‐mediated transformation of Papaver somniferum cell suspension cultures.

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes ( sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric β-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.

Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.

SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes.

In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase. In a previous study we cloned and sequenced the SAM1 gene (D. Thomas and Y. Surdin-Kerjan, J. Biol. Chem. 262:16704-16709, 1987). In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption. It has been sequenced and compared with the SAM1 gene. The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes. Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions. The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions. The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains. This raises the question of how the two AdoMet synthetases in S. cerevisiae interact to control AdoMet synthesis.

SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes.

In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase. In a previous study we cloned and sequenced the SAM1 gene (D. Thomas and Y. Surdin-Kerjan, J. Biol. Chem. 262:16704-16709, 1987). In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption. It has been sequenced and compared with the SAM1 gene. The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes. Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions. The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions. The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains. This raises the question of how the two AdoMet synthetases in S. cerevisiae interact to control AdoMet synthesis.