Quantitative expression of representative GACA/GATA-tagged transcripts demonstrating variations among somatic/gonadal tissues and spermatozoa

Four types of expressional profiles were uncovered with GACA; some transcripts with highest expression in testis and spermatozoa e.g. Ankyrin repeat domain , few in testis only e.g. Ubap1 , few in spermatozoa only e.g. novel pJSC3 , and others distributed almost uniformly in all the tissues e.g. HBGF-1 . Three types of expressional profiles were observed for GATA-tagged transcripts; some showed highest expression both in testis and spermatozoa e.g. novel pJSC34 , few in testis only e.g. novel pJSC33 , few others in spermatozoa only e.g. novel pJSC32 , and others highest in testis and spermatozoa but with minimal variation in comparison to somatic tissues e.g. novel pJSC31 . For details, see table 3 and text.<p><b>Copyright information:</b></p><p>Taken from "Organization and differential expression of the GACA/GATA tagged somatic and spermatozoal transcriptomes in Buffalo "</p><p>http://www.biomedcentral.com/1471-2164/9/132</p><p>BMC Genomics 2008;9():132-132.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2346481.</p><p></p

Primers used for Site directed Mutagenesis.

<p>Primers used for Site directed Mutagenesis.</p

Active site interactions of EhSAT1.

<p>Image showing interaction of A) L-ser and B) L-Cys with the active site residues of EhSAT1 as visualized in crystal structure of EhSAT1-ser (PDB id 3Q1X) and EhSAT1-cys (PDB id 3P47) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055932#pone.0055932-Nozaki1" target="_blank">[8]</a>. A) A salt bridge is formed in between the carboxyl group of Ser and the side chain of Arg 222A. This carboxyl group also makes a hydrogen bond with side chain of His 208A and a water molecule. Side chains of His 223A and His 180B make other hydrogen bonds with the hydroxyl group of the serine. The amino group of serine forms a salt bridge with the carboxyl group of the Asp 114B and Asp 179B. B) Interactions with the inhibitor L-cysteine bound at active site of EhSAT1. Cysteine coordinates with the same residues as that of serine, but with minor repositioning. The carboxyl oxygen interacts with the amide group of the Arg 222A and also with His 223A and His 208A. The thiol group interacts with His 180B, His 223A, and a water molecule. The amine of cysteine bonds with the carboxyl groups of the Asp 114B and Asp 179B. The image was prepared using Pymol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055932#pone.0055932-Schrodinger1" target="_blank">[26]</a>.</p

Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from <em>Entamoeba histolytica</em> Assists in Partial Regaining of Feedback Inhibition by Cysteine

<div><p>The cysteine biosynthetic pathway is essential for survival of the protist pathogen <i>Entamoeba histolytica,</i> and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated <i>in vitro</i>, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K_m, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.</p> </div

The percent of SAT activity remaining in the presence of increasing concentrations of cysteine.

<p>Reactions were carried out in the presence of 3 mM serine. Native EhSAT1 was inhibited completely by 0.1 mM cysteine while the H208S-EhSAT1 mutant was inhibited by only about 85% even at a concentration of 2 mM Cys. As expected, native EhSAT3 was not inhibited by cysteine but the S208H-EHSAT3 showed about 36% inhibition.</p

Proponderance of CpG dinucleotides in the mammalian nullomers of length 11 base pairs.

<p>Note.–The 11 base pair nullomer sequences were obtained from web resource <a href="http://trac.boisestate.edu/dna/applets/SeqCount.html" target="_blank">http://trac.boisestate.edu/dna/applets/SeqCount.html</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001022#pone.0001022-Hampikian1" target="_blank">[1]</a>).</p

Kinetic study of EhSAT3.

<p>Michaelis Menten representation of velocity values plotted against the serine concentrations for EhSAT3 native and mutant (S208H). Kinetic studies were done in 50 mM Tris buffer pH 8.0 keeping acetyl CoA concentration constant at 0.1 mM and varying concentration of serine from 10 µM to 500 µM. The inhibition studies were done in presence of 5 µM and 10 µM of cysteine. K_m was calculated by the Michaelis Menten equation using Sigma Plot software. Standard deviations are calculated from the three independent experiments for each substrate concentration values. EhSAT3 activity was not affected by the mutation but the mutant EhSAT3 show increased sensitiveness to cysteine inhibition.</p

The binding free energy of EhSAT1 and EhSAT3 complexes.

<p>The table shows the detailed contribution of energy components calculated using Poisson Boltzmann Surface Area (MM-PBSA) method for EhSAT1, EhSAT3 and their mutants to evaluate their binding activity. Here ΔE_Ele, electrostatic interactions; ΔE_Vdw, van der Waals interactions, ΔE_MM = ΔE _Ele+ΔE_Vdw, ΔG_sol-ele: polar solvation free energy are calculated by solving the Poisson-Boltzmann equation PB; ΔG_sol-np, non-polar solvation free energy, ΔG_polar = ΔE _Ele+ΔG_sol-ele; ΔG_nonpolar = ΔE_Vdw+ΔG_sol-np, ΔG_Bind = estimated total binding free energy.</p

RT-PCR analyses for representative GACA- and GATA- tagged transcripts using internal primers and cDNA from different somatic tissues, gonads and spermatozoa as templates

The transcript IDs are given on the left and names of the tissues on the top. Quality and quantity of the cDNA samples was normalized and genomic contamination in the RNA checked by PCR with β-actin derived primers. Tissue specificities of the transcripts were ascertained on the basis of presence or absence of amplicons using the respective cDNA templates which were further confirmed by real time PCR and Southern blotting.<p><b>Copyright information:</b></p><p>Taken from "Organization and differential expression of the GACA/GATA tagged somatic and spermatozoal transcriptomes in Buffalo "</p><p>http://www.biomedcentral.com/1471-2164/9/132</p><p>BMC Genomics 2008;9():132-132.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2346481.</p><p></p

Protein sequence alignment of EhSAT1 and EhSAT3.

<p>Protein sequence alignment was done using ClustalW alignment program. Conserved active site residues are highlighted in yellow boxes while the active site residues (at position 208) that differ in the isoforms are highlighted in a pink box.</p