Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5 promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5 up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

Effects of 20-OH-ecdysone on Drosophila cells: Regulation of endogenous and transfected genes

Drosophila cell lines respond to physiological doses of 20-OH-ecdysone by entering mitotic arrest and differentiating morphologically. The cells also exhibit changes in gene expression. Several enzyme activities are induced, and the synthesis of cytoplasmic actin and of the four small heat-shock proteins (hsp) is initiated. Hybrid genes, containing the 5′ region of Drosophila heat-shock protein genes ligated to the herpes simplex virus thymidine kinase gene (tk), have been transfected into cells of the Drosophila cell line S3. Constructions containing sequences upstream from hsp 70, or from any of the small hsp genes, show heat-inducible tk expression. Ecdysterone-inducible tk expression is seen only in transfections with small hsp-tk hybrid genes. This transient expression system can be used as an assay for function to define regions of DNA, flanking the coding region of inducible genes, which are necessary for normal gene expression and gene regulation in cultured cells.     

A developmental study of acid phosphatase-1 in Drosophila melanogaster.

The developmental profile of acid phosphatase-1 activity in Drosophila melanogaster indicates that this lysosomal gene-enzyme system (Acph-1, 3–101.1) is responsible for ca. 90% of the low-pH nucleotidase activity throughout development. The enzyme is present at particularly high levels during embryogenesis. It is shown with electrophoretic variants and null mutants of acid phosphatase-1 that virtually all of the embryonic enzyme is maternal in origin and is made during oogenesis. The enzyme exists in several isozymic forms at fertilization, and all but one of these forms disappear during early embryogenesis. Detectable maternal enzyme persists until the third larval instar stage. Crosses between females homozygous for a null allele and wild-type males show the zygotic Acph-1 gene activation occurs by at least 9 hr after oviposition.     

Molecular characterization of a venom acid phosphatase Acph-1-like protein from the Asiatic honeybee Apis cerana

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45 °C and pH 4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.     

A Gene Located at 56F1-2 inDrosophila melanogasterEncodes a Novel Metazoan β-like Subunit of Casein Kinase II

Drosophila melanogastercasein kinase II (DmCKII) is composed of catalytic α and regulatory β subunits associated as an α₂β₂heterotetramer. Using the two-hybrid system, we have screened aDrosophilaembryo cDNA library for proteins that interact with DmCKII α. One of the cDNAs encodes a novel β-like polypeptide, which we designate β′.In situhybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the β gene and theStellatefamily of β-like sequences. The predicted sequence of β′ is more closely related to the β subunit ofDrosophilaand other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit.In vitroreconstitution studies show that a GST-β′ fusion protein associates with the α subunit to generate a tetrameric complex with regulatory properties similar to those of the native α₂β₂holoenzyme. The data are consistent with the proposed role of the β′ subunit as an integral component of the holoenzyme.     

Localization at the ultrastructural level of maternality derived enzyme and determination of the time of paternal gene expression for acid phosphatase-1 in Drosophila melanogaster.

Ultrastructural histochemistry instead of acrylamide gel electrophoresis (see R. Yasbin, J. Sawicki, and R. J. MacIntyre, 1978, Develop. Biol. 63, 00-00) is used to determine the time of paternal gene expression for the enzyme acid phosphatase-1 of Drosophila melanogaster in $\text{Acph-1}{}^{\mathrm{n}}\text{Acph-1}{}^{\mathrm{B}}$ embryos in which the null allele is derived from the female parent. Timed embryos were histochemically stained for acid phosphatase activity according to the lead phosphate method of Gomori and were examined at the ultrastructural level. Enzyme activity, resulting from activation of the paternal Acph-1 gene, is detected as early as 5 hr after fertilization. Maternally derived enzyme in $\text{Acph-1}{}^{\mathrm{B}}\text{Acph-1}{}^{\mathrm{B}}$ embryos is found principally in the yolk regions and around invaginations. This suggests that acid phosphatase-1 functions in yolk digestion and in cell movements during early embryogenesis.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization,evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5′ border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3′ acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue

A cDNA which encodes a calnexin (Cnx)-like protein from Drosophila melanogaster has been characterized. The deduced amino acid sequence shares several regions of homology with Cnx from other sources with two conserved motifs each repeated four times. The gene was found to be transcribed in various tissues and at all developmental stages. We have mapped the gene at chromosomal position 99A and we have also mapped the related gene coding for Drosophila calreticulin at 85E.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5′CYCGRG3′ and cleave between the first C and second Y to generate a four-base 5′ extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the “endo-blue” method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Molecular cloning of the murine branched chain α-ketoacid dehydrogenase E2 subunit: presence of 3′ B1 repeat elements

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain α-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5′ sequence encoding the amino-terminal preprotein sequence and 3′ untranslated region are less well conserved. The 3′-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3′-noncoding region of human E2 cDNA.     

A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins,wasp venom antigen 5, and plant group 1 pathogenesis-related proteins

The CAP protein family is made up of a group of secreted proteins that share sequence similarity. Members of this family are found in animals, plants, and fungi, and their shared sequence similarity suggests that members share a common, but as yet unknown, molecular function. As a first step in defining the function of CAP family proteins, an 878 bp partial cDNA encoding a novel member of the CAP family was cloned by the polymerase chain reaction (PCR) from total RNA of adult Drosophila. The cDNA contained the complete coding sequence for a protein 256 amino acids in length, as well as the complete 3′ untranslated region (UTR) and a portion of the 5′ UTR. The protein, named Antigen 5-related (Agr), was most similar in sequence to antigen 5 (Ag5), a CAP family member found in social wasps and ants. The corresponding Agr RNA is about 1 kb in length and is present at all stages of development, with highest levels observed in adults. Agr RNA is transcribed from a single gene that is located within region 12F of the X chromosome. The identification of Agr in Drosophila expands the number of known CAP family members to well over four dozen. Further studies of Agr and the gene which encodes this protein using the Drosophila model system may help provide important insight into the molecular functioning of this little known, but increasingly significant protein family.     

Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases.

Molecular cloning of a full-length cDNA for human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to previously described enteric acid lipases, but lacks significant homology with any characterized neutral lipases. The lysosomal enzyme catalyzes the deacylation of triacylglyceryl and cholesteryl ester core lipids of endocytosed low density lipoproteins; this activity is deficient in patients with Wolman disease and cholesteryl ester storage disease. Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites. Transfection of the cDNA into Cos-1 cells resulted in the expression of acid lipase activity with the substrate range of the native enzyme at a level that was greater than 40 times the endogenous activity.     

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.     

西伯利亚蓼rd22基因的克隆与序列分析

以3% NaHCO₃溶液胁迫处理48 h的西伯利亚蓼为试材,利用RACE技术,从其茎部组织克隆了脱水应答蛋白RD22的全长cDNA序列。测序后的结果分析表明,该cDNA序列全长为1 302 bp,5′非翻译区为59 bp,3′非翻译区为25 bp,开放读码框为1 218 bp,编码405个氨基酸。在氨基酸序列的C端含有一个比较保守的BURP结构域,N端含有5个重复序列THV-VGKGGV-V。信号肽检测证明该蛋白为分泌性蛋白,前21个氨基酸区域为信号肽结构。其推演的氨基酸序列与葡萄的同源性最高,达到60%。该基因已在GenBank上注册,基因序列登录号为DQ836050。     

Cloning and sequencing of a copper-containing,topa quinone-containing monoamine oxidase from human placenta

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3′-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)₅ tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a M_r of 82 525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.