TATA Box Research Articles

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

BackgroundIn biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.ResultsIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.ConclusionWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

UGT1A1 Polymorphism and Mortality Outcome after Allogeneic Stem Cell Transplantation with Busulfan Based Regimen

Introduction: Mutations in the UGT1A1 gene have been implicated in Gilbert's syndrome, which shows mild hyperbilirubinemia, and a more aggressive childhood subtype, Crigler-Najjar syndrome. A common cause of decreased UGT1A1 activity is the insertion of a TA in the TATA box at the promoter region of the UGT1A1 gene which was named as UGT1A1*28 . Individuals with homozygous UGT1A1*28 had higher levels of serum bilirubin compared with those with heterozygous UGT1A1*28 or the wild-type allele.Gilbert's syndrome, also known as constitutional hepatic dysfunction or familial nonhemolytic jaundice, is an inherited disorder of the liver resulting in an overabundance of bilirubin. Most of the patients with Gilbert's syndrome are asymptomatic; however, they sometimes present with episodes of mild intermittent jaundice due to predominantly unconjugated hyperbilirubinemia.Emerging data on the role of genetic variants in the UGT1A1 gene confirm that the UGT1A1*28 allele is associated with severe toxicities in irinotecan-based chemotherapy.Busulfan is an alkylating drug with a narrow therapeutic index which can cause toxicity with higher plasma levels. Recently Mc Donald et al demonstrated in a large cohort of patients that overall mortality and non relapse mortality at 200d after hematopoietic stem cell transplantation were significantly higher in patients with Gilbert's syndrome receiving busulfan-containing conditioning regimens as compared to patients without Gilbert's syndrome without analysis of genomic variants affecting the function of UGT1A1.In a retrospective study we analyzed the role of UGT1A1*28 polymorphism on the transplant related mortality in a cohort of patients submitted to allogeneic stem cell transplantation conditioned with a busulfan-based regimen.Patients: We evaluated 74 patients (M/F 46/28), median age 48ys (range 21-66), underlying diseases were AML (50 pts), ALL (5 pts), MDS (10 pts), NHL (3 pts), CLL (1 pt), MPN (1 pt) and MM (2 pts). Conditioning regimen used were: BuCy (23 pts), BuFlu (32 pts), TBF (17 pts) and BuMel (2 pts).Results: We found 19 wild type (WT) pts (25%), 31 heterozygous (41%) and 24 homozygous pts (32%). Bilirubin levels were significantly higher in homozygous patients as compared to heterozygous and normal both at baseline and during the early follow-up post-transplant (p<0.001 and p=0.01 respectively) (Fig.1). When we looked at correlation between polymorphism and TRM at 200d and at 3ys after transplant it was 15.7% and 17.0% in WT pts, 6.4% and 26.6% in the heterozygous group and 20.8% and 27.3% in the homozygous group respectively (not statistically different).Conclusions: We demonstrated that patients homozygous for UGT1A1 polymorphism have higher levels of total bilirubin during transplant as compared to WT and heterozygotes but we did not find any correlation between TRM in patients receiving busulfan as part of the conditioning regimen for allo SCT and UGT1A1 polymorphism. Despite this result we are testing the polymorphism on a larger and homogenous cohort of patients because in our opinion it could be useful to identify patients with this genetic mutation responsible of high bilirubin levels at transplant which could be wrongly considered a criteria for diagnosis of VOD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells.

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci. 2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

GENOTYPING OF CAMELS OF ALMATY REGION BY CSN2 DAIRY PRODUCTIVITY GENE

Camel milk is highly valued for its medicinal properties and high nutritional value. Camel milk and its products are easily digestible and therefore natural for the human body. Thus, the study of genetic diversity and selection of camels according to preferred genotypes is an important component of camel breeding in Kazakhstan. The β-casein protein, encoded by the CSN2 gene, makes up the largest part of the casein fraction of camel milk and is considered the main gene for the presence of alleles associated with different levels of expression. The g.2126A> G transition is located the TATA box region of the CSN2 gene promoter, with a presumed effect on the transcription factor binding activity, which determines the intensity of this gene expression. In the studied camel populations bred in the Almaty region, the total number of which is 53 animals, the frequency of the G allele was 0.36, while the frequency of the A allele was 0.64. Also, the Hardy-Weinberg equilibrium was equal to 0,006. The level of polymorphism showed a high result in populations 1, 2 and 4, amounting to 100%, while in the third population this indicator was 66.67%. For the studied populations, such statistical indicators were identified as the genotypes occurrence frequency, the observed and effective number of alleles, Nei's gene diversity etc., which give the population genetic characteristics of populations.

Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences.

Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.

Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes.

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

Mastering the control of the Rho transcription factor for biotechnological applications

The present review represents an update on the fundamental role played by the Rho factor, which facilitates the process of Rho-dependent transcription termination in the prokaryotic world; it also provides a summary of relevant mutations in the Rho factor and the insights they provide into the functions carried out by this protein. Furthermore, a section is dedicated to the putative future use of Rho (the 'taming' of Rho) to facilitate biotechnological processes and adapt them to different technological contexts. Novel bacterial strains can be designed, containing mutations in the rho gene, that are better suited for different biotechnological applications. This process can obtain novel microbial strains that are adapted to lower temperatures of fermentation, shorter production times, exhibit better nutrient utilization, or display other traits that are beneficial in productive Biotechnology. Additional important issues reviewed here include epistasis, the design of TATA boxes, the role of small RNAs, and the manipulation of clathrin-mediated endocytosis, by some pathogenic bacteria, to invade eukaryotic cells. KEY POINTS: • It is postulated that controlling the action of the prokaryotic Rho factor could generate major biotechnological improvements, such as an increase in bacterial productivity or a reduction of the microbial-specific growth rate. • The review also evaluates the putative impact of epistatic mechanisms on Biotechnology, both as possible responsible for unexpected failures in gene cloning and more important for the genesis of new strains for biotechnological applications • The use of clathrin-coated vesicles by intracellular bacterial microorganisms is included too and proposed as a putative delivery mechanism, for drugs and vaccines.

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Assembling for transcription initiation

Transcription Eukaryotic transcription initiation by RNA polymerase II (Pol II) requires the assembly of a preinitiation complex (PIC) on core promoters. The binding of TATA box–binding protein (TBP) to the TATA box promoter has been thought to be a general rule in PIC assembly and transcription initiation. However, most coding genes lack a TATA box, and nearly all Pol II–mediated gene transcription requires the TBP-containing multisubunit complex transcription factor IID (TFIID). Chen et al. determined the structures of human TFIID-based PIC in sequential assembly states and revealed that TFIID supports distinct PIC assembly on TATA-containing and TATA-lacking promoters. The finding resolves the long-standing mystery of how one set of general transcription machinery initiates transcription on diverse promoters. Science , this issue p. [eaba8490][1] [1]: /lookup/doi/10.1126/science.aba8490

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (PolII) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus PolII, which is 99.9% identical to human PolII. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8Å resolution, and resolve the structure of TFIIH at 2.9-4.0Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Structural insights into preinitiation complex assembly on core promoters.

Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters). The two tracks converge at an ~50-subunit holo PIC in identical conformation, whereby TFIID stabilizes PIC organization and supports loading of cyclin-dependent kinase (CDK)-activating kinase (CAK) onto Pol II and CAK-mediated phosphorylation of the Pol II carboxyl-terminal domain. Unexpectedly, TBP of TFIID similarly bends TATA box and TATA-less promoters in PIC. Our study provides structural visualization of stepwise PIC assembly on highly diversified promoters.

Identification of nucleotide variation of growth hormone gene in rabbit populations reared in Bulgaria

Five rabbit populations of New Zealand White (NZW), Californian (CAL), crossbred NZW×GW and two generations of the synthetic population – SPF&lt;sub&gt;1&lt;/sub&gt; and SPF&lt;sub&gt;2&lt;/sub&gt; reared in Bulgaria were included in the present study with the aim of detecting the genetic variability of the growth hormone encoding gene (&lt;em&gt;GH&lt;/em&gt;) via polymerase chain reaction with the restriction fragment length polymorphism analysis and direct sequencing. The targeted region of the rabbit &lt;em&gt;GH&lt;/em&gt; gene was amplified and a fragment of a total of 231 bp was obtained in all studied populations. Allele identification was determined after enzymatic digestion, where two fragments of 62 and 169 bp correspond to allele C and an undigested fragment of 231 bp corresponds to allele T. Two additional bands of 107 and 124 bp evidenced A/G genetic polymorphism in the rabbit &lt;em&gt;GH&lt;/em&gt; gene. Thirtyeight percent of the studied rabbits were carriers of the double mutation (C/T+A/G) in the same locus as the studied &lt;em&gt;GH&lt;/em&gt; gene. The sequence analysis revealed two nucleotide substitutions – g.111C&amp;gt;T and g.156A&amp;gt;G in the non-coding region between the regulatory TATA box and 5’ UTR region, and a novel g.255G&amp;gt;A genetic variant in intron 1 of GH gene. The A&amp;gt;G transition was most frequent (40.57%), compared to the other ones, G&amp;gt;A (28.57%) and C&amp;gt;T (10.80%), respectively. The most frequent genotype in the NZW population was homozygous TT (0.93), with a prevalence of the T allele (0.97) over allele C (0.03) for g.111C&amp;gt;T SNP site. The distribution of the allele and genotype frequencies at the sites g.156A&amp;gt;G and g.255G&amp;gt;A in this rabbit group was identical, with the highest value of 0.93 for alleles A and G, respectively. The rabbit populations CAL and NZW×GW showed equal frequencies of the prevalent T allele (0.83) and for homozygous TT genotype (0.67) according to g.111C&amp;gt;T SNP. The highest values were obtained for the allele А (0.83) and for homozygous AA genotype (0.67) at c.33A&amp;gt;G SNP in these rabbit groups. The highest values (0.67, 0.60 and 0.80) for the heterozygous genotypes at g.111C&amp;gt;T, g.156A&amp;gt;G and g.255G&amp;gt;A SNPs, respectively, were detected among the SPF&lt;sub&gt;2&lt;/sub&gt; rabbit population, compared to the both homozygous genotypes. The results obtained in the present research indicates a significant degree of genetic variability of the studied polymorphic &lt;em&gt;GH&lt;/em&gt; locus in the SPF&lt;sub&gt;2&lt;/sub&gt; rabbit group.

Cloning, characterization, and transcriptional activity of β-actin promoter of African catfish (Clarias gariepinus).

Selection of suitable promoters is crucial for the efficient expression of exogenous genes in transgenic animals. Although one of the most effective promoters, the β-actin promoter, has been widely studied in fish species, it still remains unknown in the economical important African catfish (Clarias gariepinus). In this study, the β-actin promoter of African catfish (cgβ-actinP) was cloned and characterized. In addition, recombinant plasmid pcgβ-actinP-EGFP with enhanced green fluorescent protein (GFP) gene as the reporter gene was constructed to verify the transcriptional activity. We obtained a cgβ-actinP fragment length of 1405bp, consisting 104bp of the 5' proximal promoter, 96bp of the first exon, and 1205bp of the first intron. Similar to those of other fish species, cgβ-actinP contains three key transcription regulatory elements (CAAT box, CArG motif, and TATA box). GFP-specific fluorescent signals were detected in chicken embryonic fibroblasts cells (DF-1 cells) transfected with pcgβ-actinP-EGFP, which was approximately 1.11 times of the positive control. In addition, GFP was effectively expressed in zebrafish larvae microinjected with linearized cgβ-actinP-EGFP, with expression rate reaching approximately 49.84%. Our data indicate that cgβ-actinP could be a potential candidate promoter in the practice of constructing "all fish" transgenic fish.

Identifying UGT1A1 gene mutations in Vietnamese patients with Gilbert syndrome

Gilbert’s syndrome (GS) is the most common inherited disorder of bilirubin metabolism, non-lethal, affecting 3-12% of the population. The genetic variants of the UDP- glucuronosyltransferase 1A1 (UGT1A1) gene might reduce the gene transcription activity and its enzyme expression, which affects the ability to conjugate glucuronidation in the liver. This study aimed to identify genetic variants of UGT1A1 in two Vietnamese sibling brothers with jaundice manifestations suspected GS. The peripheral blood samples of patients were used to extract genome DNA and sequence the enhancer, promoter, and coding all five exons of UGT1A1. Two pathogenic variants c.-3279T&gt;G located in the phenobarbital responsive enhancer module (gtPBREM) and A(TA)7TAA of the TATA box in the promoter region were identified. They are twice common pathogenic variants that were reported in almost hyperbilirubinemia individuals from different populations. The obtained results improved the accuracy of medical diagnosis and warned the patients to be cautious in case they have to use medical drugs in the future.

Gilbert's Syndrome, Bilirubin Level and UGT1A1∗28 Genotype in Men of North-West Region of Russia

Gilbert's syndrome (GS) is a hereditary pathology that affects approximately 10% of the world's population. In most cases, GS is associated with the UGT1A1∗28 polymorphism of UGT1A1 gene coding the enzyme bilirubin uridine diphosphate glucuronosyltransferase (UGT-1A) which plays a key role in the bilirubin metabolism. The presence of an additional TA repeat in the TATA box of the UGT1A1 gene promoter (the allelic variant of 7TA, abbreviated as UGT1A1∗28) leads to a significant decrease in the enzymatic activity of UGT-1A in the liver and to decrease in glucuronidation process as a consequence. The aim of the study is to estimate the prevalence of the 6TA/6TA, 6TA/7TA, and 7TA/7TA genotypes of UGT1A1 promoter and to analyze the effect of these variants on bilirubin levels in healthy men in North-West Russia and patients with a clinical diagnosis of GS. Genotyping of the UGT1A1 ∗28 (rs8175347) polymorphism was carried out by real-time PCR. The results obtained indicate an increased probability of GS developing in residents of the North-West region of Russia compared with other representatives of the Caucasians. Despite the fact that the level of serum bilirubin increases with the rise in the number of additional TA dinucleotides in the UGT1A1 gene promoter tests of clinical manifestations only (jaundice, fatigue, sleep disturbances, nausea, belching, and so on) and increased bilirubin levels in patients with normal liver function do not allow unequivocally diagnose GS. UGT1A1∗28 genotyping should be used as a prognostic risk factor for such pathology development.

Genomic Organization and Transcriptional Regulation of Two Homologous Fucolectin Genes AjFTL-1 and AjFTL-2 in Apostichopus japonicus

F-type lectins (Fucolectins) are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion. In our previous research, we found that two homologous Fucolectin genes, AjFTL-1 and AjFTL-2, exhibited different expression profiles after lipopolysaccharides (LPS) challenge in Apostichopus japonicus. However, the transcriptional regulation mechanism of these two genes remains largely unknown. In this study, the 5′ flanking regions of AjFTL-1 and AjFTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid (EPC) cell system. First, in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB, CREB, and CREBP1, and both contain a TATA box. Additionally, luciferase assay and progressive 5′ truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells. Moreover, the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan (PGN) and mannan (MAN), while AjFTL-2 promoter was prominently regulated by LPS and MAN, indicating AjFTL-1 and AjFTL-2 genes showed different transcriptional regulation pattern under different immune stimulation. More importantly, analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites (−769 bp to −761 bp, −185 bp to −172bp) in AjFTL-1 and one potential binding site (−530 to −517bp) in AjFTL-2. Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity, but not AjFTL-1 promoter activity. These findings indicated that in marine invertebrates, different Fucolectin members differ in transcription regulations and expression patterns, and might play different roles in immune defenses during pathogen infection.