Association of 25 bp deletion in MYBPC3 gene with left ventricle dysfunction in coronary artery disease patients

Rationale

Mutations in MYBPC3 encoding cardiac myosin binding protein C are common genetic cause of hereditary cardiac myopathies. An intronic 25-bp deletion in MYBPC3 at 3′ region is associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies in Southeast Asia. However, the frequency of MYBPC3 25 bp deletion and associated clinical presentation has not been established in an unrelated cohort of left ventricular dysfunction (LVD) secondary to coronary artery disease (CAD) patients.

Objective

We sought to determine the role of MYBPC3 25 bp polymorphism on LVD in two cohorts of CAD patients.

Methods and Results

The study included 265 consecutive patients with angiographically confirmed CAD and 220 controls. MYBPC3 25 bp polymorphism was determined by polymerase chain reaction. Our results showed that carrier status of MYBPC3 25 bp deletion was associated with significant compromised left ventricle ejection fraction (LVEF ≤45) in CAD patients (p value  =  <0.001; OR = 4.49). To validate our results, we performed a replication study in additional 140 cases with similar clinical characteristics and results again confirmed consistent findings (p = 0.029; OR = 3.3). Also, presence of the gene deletion did not have significant association in CAD patients with preserved ejection fraction (LVEF>45) (p value  = 0.1; OR  = 2.3).

Conclusion

The frequency of MYBPC3 DW genotype and D allele was associated with compromised LVEF implying that genetic variants of MYBPC3 encoding mutant structural sarcomere protein could increase susceptibility to left ventricular dysfunction. Therefore, 25 bp deletion in MYBPC3 may represent a genetic marker for cardiac failure in CAD patients from Southeast Asia.     

Deletion / insertion polymorphisms of the prion protein gene (<Emphasis Type="BoldItalic">PRNP</Emphasis>) in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

Resistance to fatal disease bovine spongiform encephalopathy (BSE), due to misfolded prion protein in cattle, is associated with a 23-bp indel polymorphism in the putative promoter and a 12-bp indel in intron 1 of the PRNP gene. Gayal (Bos frontalis) is an important semiwild bovid species and of great conservation concern, but till today these indel polymorphisms have not been evaluated in gayals. Therefore, we collected 225 samples of gayals and evaluated the genetic indel polymorphism in the two regions of this PRNP gene. The results revealed high allelic frequencies of insertions at these indel sites: 0.909 and 0.667 for, respectively, the 23 bp and 12 bp indels, both also with significant genotype frequencies (\(\chi ^{2}\): 9.81; 23 bp and \(\chi ^{2}\): 43.56; 12 bp). At the same time, the haplotype data showed indel polymorphisms with extremely low deletion (0.01) in both regions of the PRNP gene. We compared these data with those reported for healthy and BSE-affected cattle (Bos taurus) breeds from two European countries, Germany and Switzerland, and significant difference (\(P\,{<}\,0.001\)) was observed between BSE-affected as well as the healthy cattle. Further, our data were also extensively compared with previous reports on BSE and highly significant (\(P\,{<}\,0.001\)) outcomes were observed. This result suggested negligible genetic susceptibility to BSE in gayals. To the best of our knowledge, this study is the first comprehensive deciphering information about the PRNP indel polymorphisms of 23 bp and 12 bp in gayals, a semiwild species of China.     

Molecular characterization of a novel vernalization allele <Emphasis Type="Italic">Vrn-B1d</Emphasis> and its effect on heading time in Chinese wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landrace Hongchunmai

Flowering time of wheat cultivars contributes greatly to the adaptability to environmental conditions and it is largely controlled by vernalization genes. In this study, 262 Chinese mini-core wheat cultivars were used to identify the allelic variation at VRN-B1 locus. A novel dominant allele Vrn-B1d was found in Chinese spring wheat landrace cultivar Hongchunmai. This allele contained several genetic divergence within the first intron comparing to the recessive allele vrn-B1, including one large 6850-bp deletion (670–7519 bp), one small 187-bp deletion (7851–8037 bp), one unique SNP (T to C, 7845 bp), and one 4-bp mutation (TTTT to ACAA, 7847–7850 bp). Meanwhile, it was also different from the three known dominant alleles at VRN-B1 locus. Two pairs of primers were designed to identify the novel allele Vrn-B1d and other four known alleles of VRN-B1. A multiplex PCR was established to discriminate all five alleles simultaneously. The greenhouse experiment with high temperature (non-vernalizing condition) and long light showed that F₂ plants containing Vrn-B1d allele headed significantly earlier than those with recessive vrn-B1 allele, suggesting that Vrn-B1d is a dominant allele conferring the spring growth habit. This study provides a useful germplasm and molecular markers for wheat breeding.     

Effects of Common Polymorphisms in the MTHFR and <Emphasis Type="Italic">ACE</Emphasis> Genes on Diabetic Peripheral Neuropathy Progression: a Meta-Analysis

Diabetic peripheral neuropathy (DPN) is a microvascular complication of diabetes mellitus. The aim of this meta-analysis was to evaluate the effects of methylenetetrahydrofolate reductase (MTHFR) 677 C>T and ACE I/D polymorphisms in the development of DPN. We systematically reviewed published studies on MTHFR 677 C>T and ACE I/D polymorphisms and DPN found in various types of electronic databases. Strengthening the Reporting of Observational studies in Epidemiology (STROBE) quality score systems were used to determine the quality of the articles selected for inclusion. Odds ratios (ORs) and its corresponding 95 % confidence interval (95 % CI) were calculated. We used STATA statistical software (version 12.0, Stata Corporation, College Station, TX, USA) to deal with statistical data. Our results indicated an association of ACE D>I mutation (OR?=?1.43, 95 % CI 1.12–1.83, P?=?0.004) and MTHFR 677 C>T mutation (OR?=?1.43, 95 % CI 1.08–1.90, P?=?0.014) with DPN under the allele model, and similar results were also found under the dominant model (all P?<?0.05). Subgroup analysis by country indicated that the MTHFR 677 C>T polymorphism may be the main risk factor for DPN in Turkey under four genetic models. ACE D>I mutation was correlated with DPN in Japanese and Pakistani populations in the majority of groups. The relationships of MTHFR 677 C>T and ACE I/D polymorphisms with DPN patients presented in this meta-analyses support the view that the MTHFR and ACE genes might play an important role in the development of DPN.     

Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig

Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 co-localized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding of the molecular mechanism underlying growth rate in Landrace pig breed.     

Polymorphisms of <Emphasis Type="Italic">KITLG</Emphasis>, <Emphasis Type="Italic">SPRY4</Emphasis>, and <Emphasis Type="Italic">BAK1</Emphasis> genes in patients with testicular germ cell tumors and individuals with infertility associated with AZFc deletion of the Y chromosome

Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44?19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11?12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to identify individuals with an increased risk of TGCTs.     

Association between relative bone mass and vitamin D receptor gene polymorphism

The paper reports a search for association between the relative bone tissue mass (percent of total body mass, %BT) and FokI (rs10735810), BsmI (rs1544410), and TaqαI (rs731236) vitamin D receptor gene polymorphisms. The group of apparently healthy young adults born in the central and northern regions of European Russia included 61 men and 60 women aged 16–23 years. No statistical association was detected between the FokI polymorphism and %BT. The carriers of the BsmI *A allele exhibited a higher %BT (p = 0.0172) compared to the subjects bearing the BsmI *G*G allele. The subjects with the *C*C TaqαI genotype were characterized by increased %BT compared to the carriers of the *T allele (p = 0.0018). The data are in good correspondence with the results obtained in the studies involving apparently healthy representatives of Central European and Northern European populations of the corresponding age.     

Two new complete mitochondrial genomes of <Emphasis Type="Italic">Dorcus</Emphasis> stag beetles (Coleoptera,Lucanidae)

The systematics of Dorcus MacLeay has been a long-standing debate. Mitochondrial genomes were widely used to deeply understand the phylogeny of problematic taxa in virtue of their genetic importance and comprehensiveness. To provide more useful genetic data for resolving the systematic disputation of Dorcus stag beetles. The complete mitochondrial genomes of Dorcus hopei and Dorcus seguyi were obtained using the next generation sequencing. Characteristics of the two genomes are explicated through comparing their genome organization and base composition, protein-coding genes and codon usage, intergenic spacers and non-coding region, transfer and ribosomal RNA genes and control region. Phylogenetic relationships were reconstructed using Maximum likelihood and Bayesian inference analyses based on the concatenated nucleotide sequences of 13 PCGs from 9 stag beetles and 3 scarab beetles. The complete mitogenomes of D. hopei and D. seguyi was 16,026 bp/17,955 bp long, respectively. A tandem repeat with the length of 940 bp was presented in the A+T-rich region in D. hopei. An unexpected non-coding region of 332 bp was located between nad2 and trnW in D. seguyi. The phylogenetic analyses robustly supported that D. hopei formed a branch with the generic type of D. parallelipipedus. Whereas D. seguyi was not covered in the branch of (D. hopei?+?D. parallelipipedus), but was sister to them. The results indicated that D. hopei should be a good member of Dorcus MacLeay. The taxonomic status of D. seguyi remained to be studied furtherly.     

Functional marker related to germination vigor of maize seed

Germination and synchronous seedling emergence are necessary for crop yield. One differentially expressed Cupin protein during maize seed germination had been identified in our previous study. To elucidate the functional sites in its coding gene, ZmGLP, a diverse maize association population was assayed. In alignment with B73, the ZmGLP gene contains five exons and encodes 522 amino acids, and a potential signal peptide domain from the 7 to 22 amino acids and a typical Cupin domain from the 332 to 477 amino acids were identified. The ZmGLP has a quick linkage disequilibrium decay with about 300 bp physical distance in the analyzed sequences, which implied that human selection might be undone in this gene. High genetic variations were evidenced in ZmGLP gene with 118 polymorphic sites in the association population. An Indel9 (18 bp) in the fifth exon of the intermotif region of the second Cupin domain, which influences the formation of C coil in the barrel folding, was detected significantly associated with germination vigor of maize seeds. The 18-bp insertion, which might help keeping the barrel folding intact, was a favorable allele for relative high germination vigor. Evolution analysis showed that this 18-bp deletion was a loss-of-function mutation. However, the action mode of ZmGLP is still yet to be further studied for maize improvement.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Heat shock responsiveness analysis of <Emphasis Type="Italic">Athsp70b</Emphasis> upstream region

A 1431-bp upstream fragment of Athsp70b was cloned via PCR amplification and expressed in onion epidermis by particle bombardment. Furthermore, the progressive deletions of the Athsp70b upstream fragment linked to the β-glucuronidase (GUS) coding region were performed. Then, a stable GUS expression was analyzed in tobacco BY2 cells and Arabidopsis. Our present results showed that about a 500-bp region upstream ATG of Athsp70b is suitable to confer heat inducibility to the GUS reporter gene in plants and around 116 bp contain nonperfect heat-sensitive element. This promoter responds to heat, salicylic acid, and benzyladenine. GUS staining was mainly observed in the vascular tissues and root tips, implying that Athsp70b is related to water transportation.     

Promoters pro-SmAMP1 and pro-SmAMP2 from Wild Plant <Emphasis Type="Italic">Stellaria media</Emphasis> for the Biotechnology of Dicotyledons

Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Analysis of the <Emphasis Type="Italic">Alu</Emphasis>I polymorphism in intron 1 of the human coagulation factor VIII gene: A new marker for the hemophilia a carrier detection

Frequencies of the C/T SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu?), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (C/T SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281–2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.     

Y-chromosome polymorphisms of the domestic Bactrian camel in China

Single-nucleotide polymorphisms (SNPs), microsatellites and copy number variation (CNV) were studied on the Y chromosome to understand the paternal origin and phylogenetic relationships for resource protection, rational development and utilization of the domestic Bactrian camel in China. Our sample set consisted of 94 Chinese domestic Bactrian camels from four regions (Inner Mongolia, Gansu, Qinghai and Xinjiang), we screened 29 Y-chromosome-specific loci for SNPs, analysed 40 bovine-derived microsatellite loci and measured CNVs of HSFY and SRY through Sanger sequencing, automated fluorescence-based microsatellite analysis and quantitative real-time PCR, respectively. A multicopy gene, SRY, was first found, and sequence variation was only detected in SRY in a screen of 29 loci in 13 DNA pools of individual camels. In addition, a TG repeat in the USP9Y gene was identified as the first polymorphic microsatellite in the camel Y chromosome, whereas microsatellite based on bovine sequences were not detected. The frequency of each allele varied among different populations. For the Nanjiang, Hexi and Alashan populations, a 243-bp allele was found. For the Sunite population, 241-bp, 243-bp and 247-bp alleles were detected, and the frequencies of these alleles were \(22.2\%\), \(44.5\%\) and \(33.3\%\), respectively; 241-bp and 243-bp alleles were found in other populations. Finally, CNVs in two Y-chromosomal genes were detected; CNV for HSFY and SRY ranged from 1 to 3 and from 1 to 9, respectively.     

Genome-Scale Association Study of Abnormal Scale Pattern in Yellow River Carp Identified Previously Known Causative Gene in European Mirror Carp

Common carp (Cyprinus carpio) is one of the most widely studied fish species due to its great economic value and strong environmental adaptability. Scattered scale, a typical phenotype of the mirror carp that is derived from Europe, has never been observed in the Yellow River carp previously. We recently identified approximately one fourth of the F1 progenies displaying scattered scale in a full-sib Yellow River carp family in our breeding program, despite both parents that showed wild type with normal scale patterns. This family provides us unique materials to investigate the genetic basis underlying the abnormal scale mutant in Yellow River carp population. Genome-wide association study (GWAS) and association mapping were performed based on genome-wide single nucleotide polymorphisms (SNP) genotyped with common carp 250 K SNP genotyping array in 82 samples of the Yellow River carp family. We identified a 1.4 Mb genome region that was significantly associated with abnormal scattered scale patterns. We further identified a deletion mutation in fibroblast growth factor receptor 1 a1 (fgfr1a1) gene within this genome region. Amplification and sequencing analysis of this gene revealed a 311-bp deletion in intron 10 and exon 11, which proved that fgfr1a1 could be the causal gene responsible for abnormal scattered scale in the Yellow River carp family. Since similar fragment mutation with 306-bp and 310-bp deletions had been previously reported as causal mutation of scattered scale patterns in the mirror carp, we speculate that either the deletion mutation was introduced from Europe-derived mirror carp or the deletion independently occurred in the mutation hotspot in fgfr1a1 gene. The results provided insights into the genetic basis of scale pattern mutant in Yellow River carp population, which would help us to eliminate the recessive allele of the abnormal scale patterns in Yellow River carp population by molecular marker-assisted breeding.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.