First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.     

人晶体膜内蛋白MP19基因（LIM2）启动子上一个晶体特异顺式元件的鉴定

使用重叠和变异的寡核苷酸作为探针,凝胶迁移分析和竞争实验分析了LIM2转录起始位点上游-47至-32的区域,与其高度亲和结合的一个蛋白复合体看来仅仅结合到这个DNA双链区域的“敏感”位点。这个位点的序列由4个G核苷,接着7个其他核苷酸(AACCTAA）及连着另外4个G核苷组成,即GGGGAACCTAAGGGG; 我们称其为Hsu元件。使用含有这个元件或相应的变异元件所构建的LIM2基因启动子CAT质粒的活性分析表明Hsu元件是位于LIM2基因启动子之内,它是LIM2基因表达所必须的。结合到Hsu元件的反式因子存于晶体发育期间,看来是晶体特异性的。由于LIM2基因启动子并不包含一个经典的TATA盒,这个Hsu元件可能充当RNA复制酶复合体结合的位点。     

人晶体膜内蛋白MPl9基因（LIM2）启动子上一个晶体特异顺式元件的鉴定

使用重叠和变异的寡核苷酸作为探针,凝胶迁移分析和竞争实验分析了LJM2转录起始位点上游-47至-32的区域,与其高度亲和结合的一个蛋白复合体看来仅仅结合到这个DNA双链区域的“敏感”位点。这个位点的序列由4个G核苷,接着7个其他核苷酸(AACCTAA)及连着另外4个G核苷组成,即GGGGAACCTAAGGGG;我们称其为Hsu元件。使用含有这个元件或相应的变异元件所构建的uM2基因启动子-CAT质粒的活性分析表明：Hsu元件是位于LJM2基因启动子之内,它是LJM2基因表达所必须的。结合到Hsu元件的反式因子存于晶体发育期间,看来是晶体特异性的。由于LIM2基因启动子并不包含一个经典的TATA盒,这个Hsu元件可能充当RNA复制酶复合体结合的位点。     

The Human Serum Paraoxonase/Arylesterase Gene (PON1) Is One Member of a Multigene Family

A physiological role for paraoxonase (PON1) is still uncertain, but it catalyzes the hydrolysis of toxic organophosphates. Evidence that the human genome contains twoPON1-like genes, designatedPON2andPON3,is presented here. HumanPON1andPON2each have nine exons, and the exon/intron junctions occur at equivalent positions.PON1andPON2genes are both on chromosome 7 in human and on chromosome 6 in the mouse. Turkey and chicken, like most birds, lack paraoxonase activity and are very susceptible to organophosphates. However, they have aPON-like gene with 70% identity with humanPON1, PON2,andPON3.Another unexpected finding is that the deduced amino acid sequences of PON2 in human, mouse, dog, turkey, and chicken and of human PON3 are all missing the amino acid residue 105, which is lysine in human PON1. The expanded number ofPONgenes will have important implications for future experiments designed to discover the individual functions, catalytic properties, and physiological roles of the paraoxonases.     

The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U_L) and unique short (U_S), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U_L and U_S. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U_L. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U_L, directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U_L, we concluded that our results can provide an explanation for the almost exclusive orientation of the U_L seen in packaged VZV DNA.Varicella-zoster virus ([VZV] Human Herpesvirus 3), is a highly cell-associated alphaherpesvirus that causes chicken pox (varicella) upon infection of naïve individuals (2). During primary infection, VZV is able to establish latency in cranial nerves, as well as dorsal root and autonomic ganglia, where it remains dormant until a reactivation event occurs (11). Reactivation of VZV occurs primarily in elderly or immunocompromised individuals and results in the development of shingles (herpes zoster), which is often associated with severe pain and postherpetic neuralgia (1).The VZV genome, the smallest among the human herpesviruses, is approximately 125 kbp in size and encodes at least 70 unique open reading frames (ORFs) (1). As has been reported for all alphaherpesviruses, the VZV genome consists of two unique regions, unique long (U_L) and unique short (U_S), each flanked by inverted repeat regions (TR_L, IR_L, TR_S, and IR_S) (9). In contrast to herpes simplex virus type 1 (HSV-1), the prototype alphaherpesvirus, VZV contains only very short repeats (88 bp) on either end of U_L, characteristic of members of the Varicellovirus genus (6). During alphaherpesvirus replication, four isomers of viral DNA are generated which can be distinguished by the orientation of U_L and U_S relative to each other. While all four possible isomers of HSV-1 DNA are packaged in virions as equimolar populations, virions produced by VZV and other varicelloviruses, such as equine herpesvirus type 1 (EHV-1), contain predominantly only two of the four possible isomeric forms of the genome (6, 10, 12, 15, 23). It was shown by Southern blot analysis of VZV virion DNA that inversion of the U_L region is rare and occurs in only approximately 5% of cases (6), which also may be attributed to a rare circular configuration of the genome within the virion (14). A previous report on EHV-1 suggested that inversion of the U_L region in infected cells is common but that packaging occurs in a directional manner (23). For both VZV and EHV-1, the reason for the more-or-less exclusive orientation of U_L within the virion still remains unknown.The organization of the VZV genome is similar to that of HSV-1, and over 90% of the VZV ORFs have counterparts in the HSV-1 genome (1, 13). One of the genes with a predicted HSV-1 homologue is ORFS/L, also referred to as ORF0. ORFS/L is predicted to encode a tail-anchored 157-amino-acid (aa) residue type 2 transmembrane protein and was discovered by Kemble and coworkers (13). The gene is located at the very beginning of U_L, directly adjacent to the a-like sequences that contain PacI and PacII sites crucial for the cleavage and packaging of concatameric VZV DNA (Fig. ​(Fig.1)1) (13, 20). Although no function has yet been attributed to the ORFS/L (ORF0) gene or its product, bioinformatic analysis of the VZV genome indicated that it represents a homologue of HSV-1 UL56 (RefSeq accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001348","term_id":"9625875","term_text":"NC_001348"}}NC_001348) (7, 8). While UL56 is dispensable for HSV-1 replication in vitro, it plays an important role in pathogenicity in vivo (3, 21). Little is known about the molecular mechanism of UL56 function in the case of HSV-1, but UL56 orthologues are specified by most members of the Alphaherpesvirinae subfamily (26). It was shown that the HSV-2 UL56 product localizes to the Golgi network and interacts with KIF1A, a kinesin motor protein, suggesting a role in vesicular trafficking (16, 17).Open in a separate windowFIG. 1.Overview of the VZV ORFS/L genomic region and the mutants generated. (A) Schematic representation of the VZV genome with a focus on the terminal region containing ORFS/L. Scale bars provide an accurate measure of the genome and the expanded region. (B) Overview of the mutants generated with mutations in the ORFS/L region. A cross indicates the deletion of the corresponding region. Black arrows indicate the loci of stop codon or HA tag insertion.A previous study of Kemble and coworkers also addressed the localization of the ORFS/L protein using a rabbit polyclonal antibody. It was reported that the ORFS/L product was found exclusively in the cytoplasm, which is contradictory to the findings for the HSV-2 orthologue and also to the localization of the ORFS/L protein based on in silico predictions from the primary sequence (13). ORFS/L of the P-Oka strain was recently shown to be unglycosylated but present in the virion (18). Furthermore, ORFS/L expression was detected in skin lesions of individuals, as well as neurons of dorsal root ganglia, during virus reactivation (13). In addition, the deletion of aa 29 to aa 157 of ORFS/L was shown to have an effect on viral replication in vitro and in vivo in the SCID-hu mouse model with thymus-liver implants. In this study, a virus-encoded luciferase reporter system was used to evaluate the growth properties of several bacterial artificial chromosome (BAC)-derived VZV mutants (28). However, it has remained unknown whether the observed growth defect is dependent on ORFS/L gene function or is due to the deletion of another critical sequence element.In this study, we sought to perform a systematic analysis of ORFS/L sequences. We were able to demonstrate that the ORFS/L protein localizes to the Golgi network in infected and transfected cells, providing further evidence for its predicted structure as a tail-anchored type 2 transmembrane protein and lending further support to the notion that it is the orthologue of HSV UL56. In addition, we showed that the ORFS/L gene product is important for efficient VZV replication in vitro. However, we also identified a 5′ region of the predicted ORFS/L that is essential for replication and plays a role in cleavage of viral DNA, as previously suggested by Davison and colleagues (6, 7). Since this essential region is not present at the opposite end of U_L, it could provide an explanation for the almost exclusive packaging in VZV virions of two viral DNA isomers with an invariable U_L orientation.     

人及小鼠钠通道SCN3A基因的启动子及其上游调控区的分析

为了比较研究人与小鼠SCN3A基因的启动子及其上游调控区的特征,采用5′-Full RACE方法对人及小鼠SCN3A基因的转录起始点进行了准确定位,通过序列测定及对比分析证明：确定人和小鼠SCN3A基因的转录起始点均为“A”,人SCN3A基因转录起始点位于翻译起始点上游约27 kb处,而小鼠位于翻译起始点上游约31 kb处．人SCN3A基因5′非翻译区存在两个5′非翻译外显子,而小鼠只有一个5′非翻译外显子．人和小鼠SCN3A基因核心启动子区(-80 ～+70)的同源率高达96.0%,存在相同的启动子核心元件,BRE/和TATA;在-400至+200区段内预测到人存在而小鼠不存在的转录因子有PHR1、GATA-1、FOXN2、NF-1及AP-4,小鼠存在而人不存在的转录因子Sp、Sp3及GBF．人和小鼠SCN3A启动子区特征的异同将为进一步研究该基因在人和小鼠的表达调控机制提供重要线索．     

A Novel Human Gene Encoding a G-Protein-Coupled Receptor (GPR15) Is Located on Chromosome 3

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have namedGPR15.A comparison of the amino acid sequence of the receptor encoded byGPR15with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1.GPR15was mapped to human chromosome 3q11.2–q13.1.