Correlation between RsaI restriction fragment length polymorphism and electrophoretic types of human placental alkaline phosphatase

Restriction fragment length polymorphism (RFLP) of human alkaline phosphatases was studied in a population sample from northern Sweden using a placental alkaline phosphatase (PLAP) cDNA probe. After digestion of human genomic DNA with RsaI the Southern blots showed DNA fragments most probably derived from three genes: PLAP, germ cell alkaline phosphatase (PLAP-like) and intestinal alkaline phosphatase. In agreement with a previous study, a two-allele polymorphism was found in PLAP with bands at 1.6 kilobases (A1) and 1.8 kilobases (A2). The gene frequencies of A1 and A2 were 0.46 and 0.54, respectively. There was a significant correlation between the RsaI RFLPs and electrophoretic types of PLAP; RSAI A2 showed an association with the ALP2p allele of PLAP.     

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Simultaneous Retention of Thermostability and Specific Activity in Chimeric Human Alkaline Phosphatases

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75 °C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase

FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids. We cloned and characterized the murine FATP4 gene and its cDNA. From database analysis we identified the human FATP4 genomic sequence. The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene. The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids. Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene. The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3%. Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart. Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids.     

DNA polymorphism of alkaline phosphatase isozyme genes: linkage disequilibria between placental and germ-cell alkaline phosphatase alleles.

The use of human placental alkaline phosphatase (PLAP) cDNA as a probe allows the detection and identification of restriction DNA fragments derived from three homologous genes, i.e., intestinal alkaline phosphatase (AP), germ-cell AP (GCAP), and PLAP. In previous RFLP studies we have reported linkage disequilibria between an RsaI and two PstI (a and b) polymorphic restriction sites and electrophoretic types of PLAP. In this report we present evidence that, in spite of the strong correlation with PLAP types, PstI(b) is an RFLP of GCAP. The data indicate close linkage between the PLAP and GCAP loci.     

水稻非生物胁迫响应基因OsMIP1的表达与进化分析

MID1编码R-R型的MYB转录因子,对不同的非生物胁迫均有响应,特别是在水稻(Oryza sativa)生殖期会受到干旱胁迫的诱导,进而在一定程度上可以保持花粉的育性并稳定水稻产量。为进一步研究水稻MID1对非生物胁迫的响应网络,利用酵母双杂交系统筛选出与其互作的蛋白因子OsMIP1,并利用双分子荧光互补系统在本氏烟草(Nicotiana benthamiana)细胞中得到验证。结果表明,OsMIP1编码1个预测含有ENTH/ANTH/VHS结构域的跨膜转运蛋白。OsMIP1在根、茎、叶、小穗和胚乳中均有表达。干旱胁迫下,OsMIP1在叶片和生殖器官中表达,特别是在减数分裂后的小花中表达显著上调。这些结果暗示,OsMIP1在花器官抵抗干旱胁迫中起一定的作用。在水稻营养生长阶段,OsMIP1表达还受到包括Na Cl和甘露醇在内的其它非生物胁迫的影响,暗示其可能在其它非生物胁迫调节中也具有一定的作用。植物中关于编码ENTH/ANTH/VHS结构域蛋白的研究很少。通过对MIP1亚家族进化关系进行分析,结果表明,在被子植物中,MIP1可分为6大类,这6大类分别来自被子植物祖先中原本就存在的6个拷贝,在被子植物的进化过程中又经历了多次基因重复和拷贝丢失等事件。MIP1家族成员广泛分布于被子植物中并可能具有抗胁迫等功能。     

罗伯茨绿僵菌中嘧啶前体合成酶基因MrThi12的功能研究

通过同源基因比对,在罗伯茨绿僵菌中找到了单拷贝的嘧啶前体合成酶基因MAA_02402,命名为MrThi12。该基因MrThi12全长1 234bp,cDNA序列全长1 029bp,编码342个氨基酸。构建同源重组载体,利用农杆菌介导的方法进行基因敲除。突变菌株在维生素B1缺乏的培养基上,生长很慢,菌丝形态异常,多分叉,完全不能产生气生菌丝和分生孢子。但是一旦有外源维生素B1时,生长状态能完全恢复,对家蚕的致死能力没有变化。     

Identification and characterization of the gene for Drosophila S20 ribosomal protein

A cDNA clone that encodes a Drosophila homologue of ribosomal protein S20 was isolated from a Drosophila ovary cDNA library. The Drosophila S20 gene (RpS20) is highly conserved with S20 genes in other organisms. It is a single copy gene and maps to position 92F-93A on polytene chromosomes. No Minute mutation in this location has been reported; at least five essential genes are possible candidates to encode RpS20. RpS20 message is expressed ubiquitously in embryos, but is expressed at high levels in the midgut.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

Chlorella virus SC-1A encodes at least five functional and one nonfunctional DNA methyltransferases

Chlorella virus SC-1A encodes at least six DNA methyltransferases (MTases): four N⁶-methyldeoxyadenine (m⁶A) MTases, M- CviSI (TGC^mA), M· CviSII C^mATG), M· CviSIII (TCG^mA) and M^mCviSIV (G^mATC), one 5-methyldeoxycytosine (m⁵C) MTase, M· CviSV (RC^mCG), and one nonfunctional m⁵C MTase, M· CviSVI, which is homologous to the MTase M· CviJI [RG^mC(T/C/G)] produced by another chlorella virus IL-3A. Genes encoding three of the SC-1A m⁶A MTases (M·CviSI, M· CviSII, and M· CviSIII) and the nonfunctional m⁵C MTase were cloned and sequenced. Neither M· CviSI nor M· CviSIII genes hybridized to genes for their respective isomethylomers, M· CviRI and M· CviBIII, from other chlorella viruses. However, the M· CviSII gene hybridized strongly to its M· CviAII isomethylomer gene from virus PBCV-1. Like the prototype chlorella virus PBCV-1, the SC-1A genome contains inverted terminal repeats, one of which is adjacent to the nonfunctional m⁵C MTase. The three cloned m⁶A MTase genes are distributed throughout the approx. 345 kb SC-1A genome.     

Use of differential display for the identification of touch-induced genes from an ethylene-insensitive Arabidopsis mutant and partial characterization of these genes

Touch has been shown to affect plant growth and development and ethylene has been shown to have similar effects. However, the mechanisms responsible for touch-induced responses remain unclear. Differential display PCR was used to identify touch-regulated genes from 3-week-light-grown ethylene-insensitive etr1-3 Arabidopsis (Columbia ecotype) mutant plants. The differential display PCR screening process yielded 32 cDNA fragments. Subsequent screening of the 32 fragments using northern analysis yielded three touch-inducible clones (A8A, G5A and G7F). These three cDNA were then used to screen a cDNA library. A 1.2 kb fragment for OPR3 was obtained from A8A screenings. This cDNA fragment encodes 12-oxophytodienoate-10, 11-reductase (OPR), an enzyme in the jasmonic acid biosynthetic pathway. OPR3 was found to be induced by touch, wounding, methyl jasmonate (MeJA), NaCl and CaCl₂ while ethylene and darkness had no effect. A 2 kb cDNA encoding a calcium-dependent protein kinase (CDPK32) was obtained with G5A screenings. CDPK32 was shown to be induced by touch, wounding, NaCl and darkness while ethylene and MeJA had little or no effect. A 1.4 kb cDNA encoding a novel protein was recovered from the cDNA library screenings with a G7F fragment. This cDNA had some sequence similarity to GDA1 and was designated GDL for GDA1-like cDNA. GDL was activated by touch, wounding, MeJA, NaCl and CaCl₂ while there was no induction with ethylene and darkness. Using differential display PCR we have successfully been able to identify three clones that are inducible by touch and not by ethylene.     

Structural evidence of functional divergence in human alkaline phosphatases

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family.     

Human thymosin-beta 4/6-26 gene is part of a multigene family composed of seven members located on seven different chromosomes

We have isolated a cDNA encoding the human interferon-inducible gene 6-26, by screening a cDNA library with an oligodeoxynucleotide probe. Its sequence was found to be identical to that of the human thymosin-beta 4 cDNA, which encodes a protein present in most cell types, but whose function is not clear at present. By hybridization of the thymosin-beta 4/6-26 cDNA to the DNA of a panel of human-rodent somatic cell hybrids, we found that at least seven genes homologous to this cDNA are present in the human genome. We localized these genes, some of which might be pseudogenes, to seven distinct chromosomes, namely, chromosomes 1, 2, 4, 9, 11, 20, and X.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.