Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants.

Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester. The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined. The proteinase was purified to homogeneity from the soluble fraction of an overproducing strain possessing the cloned gene. N-terminal amino acid sequencing of the purified protein revealed that the cloned gene is the structural gene for the protein, with the protein being synthesized in precursor form with a signal peptide. On the basis of its molecular mass (20 kDa), periplasmic localization, and substrate specificity, we conclude this protein to be protease I. By using the gene cloned on a plasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene (Kmr) on the chromosome. The Kmr gene was mapped at 11.8 min, the gene order being dnaZ-adk-ush-Kmr-purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonella typhimurium. Therefore, the gene was named apeA. Deletion of the apeA gene, either with or without deletion of other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the various media tested.     

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.     

Primary structure of the potato virus X genome: the region preceding the capsid protein cistron

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.     

Molecular characterization of the B' regulatory subunit gene family of Arabidopsis protein phosphatase 2A.

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants. We have identified a novel Arabidopsis gene (AtB' delta) which encodes a 55-kDa B' type regulatory subunit of PP2A. The protein encoded by this gene is 57-63% identical and 69-74% similar to the previously identified AtB' genes. The AtB' delta gene appears to be expressed in all Arabidopsis organs indicating its protein product has a basic housekeeping function in plant cells. Unlike certain mRNAs derived from the AtB' gamma gene, AtB' delta mRNAs do not fluctuate significantly in response to heat stress. Further analysis of cDNA sequences derived from the AtB' genes identified an alternatively spliced cDNA derived from AtB' gamma. This cDNA differs from the previously identified AtB' gamma cDNA by the absence of a 133-bp region in its 5' untranslated region. The missing 133-bp region appears to constitute an unspliced intron and its presence in the AtB' gamma gene was confirmed by PCR using Arabidopsis genomic DNA as a template. AtB' gamma mRNA containing the 133-bp intron accumulate in all Arabidopsis organs and their levels fluctuate differentially in response to heat stress. The 133-bp insert contains two short open reading frames and hence might serve as a translational control mechanism affecting AtB' gamma protein synthesis. Finally we show, using both the yeast two hybrid system and in vitro binding assays, that the B' subunit of Arabidopsis PP2A is able to associate with other PP2A subunits, supporting the notion that the B' protein serves as a regulator of PP2A activity in plants.     

Nuclear proteins from lactating mammary glands bind to the promoter of a milk protein gene.

The gene for the whey acidic protein (WAP) is expressed specifically in the lactating mammary glands of rodents. We present evidence that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the WAP gene promoter/upstream region. As monitored by mobility shifts, nuclear proteins from lactating mammary glands and from the mammary cell line MCF-7 form four high affinity complexes with a fragment spanning the region between nucleotides -175 and -88. Nuclear proteins from liver and HeLa cells generate only three high affinity complexes. DNAaseI and ExonucleaseIII protection confirmed the binding of mammary nuclear proteins to specific sequences in the WAP gene upstream region. This is the first report to describe the interaction of nuclear proteins from lactating mammary glands with cognate binding sites in the promoter/upstream region of a milk protein gene. The possibility of the binding sites being candidates for cis-acting regulatory elements governing the regulated expression of the WAP gene is discussed.     

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.