Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.     

Characterization of a cDNA encoding a novel plant poly(A) polymerase

We have isolated cDNA clones encoding a novel factor (PAP-I) that is a component of a multi-subunit poly(A) polymerase from pea seedlings. The encoded protein, when isolated from appropriately engineered Escherichia coli, was active as a poly(A) polymerase, either with an associated RNA binding cofactor (PAP-III) or with free poly(A) as an RNA substrate. The latter observation indicates that PAP-I is in fact a poly(A) polymerase. PAP-I bore a striking resemblance to an as yet uncharacterized cyanobacterial protein. This observation suggested a possible chloroplast localization for PAP-I. This hypothesis was tested and found to be substantiated; immunoblot analysis identified PAP-I in chloroplast but not nuclear extracts. Our results suggest that PAP-I is a component of the machinery that adds poly(A) to chloroplast RNAs.     

A human mitochondrial ferritin encoded by an intronless gene

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.     

A novel nuclear human poly(A) polymerase (PAP), PAP gamma.

Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. Here we show that the 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAP gamma (hsPAP gamma). hsPAP gamma shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAP gamma exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAP gamma and hsPAPII have similar organization of structural and functional domains. hsPAP gamma contains a U1A protein-interacting region in its C terminus, and PAP gamma activity can be inhibited, as hsPAPII, by the U1A protein. hsPAPgamma is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on this and previous studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAP gamma establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms.     

Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system.

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.     

Induction of poly(ADP-ribose) polymerase gene expression in lectin-stimulated human T lymphocytes is dependent on protein synthesis

The poly(ADP-ribose) polymerase mRNA level in quiescent T lymphocytes was low, but was significantly higher than that in B lymphocytes or monocytes. When T lymphocytes were stimulated with phytohemagglutinin, a prompt increase in the mRNA level was observed from 4 hours after stimulation. The level of poly(ADP-ribose) polymerase mRNA reached a maximum in the late G1 phase about 1-2 days after lectin stimulation, and then decreased gradually returning to the basal level 10 days after lectin stimulation. Cycloheximide abrogated increase in poly(ADP-ribose) polymerase gene expression suggesting that a newly synthesized protein(s) was involved in poly(ADP-ribose) polymerase gene induction in lectin-stimulated T lymphocytes.     

Testis-specific expression of a novel mouse defensin-like gene,Tdl

In order to isolate genes regulating sex differentiation of embryonic gonads, we have attempted to obtain genes specifically expressed in male embryonic gonads by means of subtractive hybridization screening, and we have cloned a novel mouse gene, Tdl. It potentially encodes a protein showing sequence homology to anti-microbial protein, beta-defensins. Tdl reveals structural features such as the six cysteine residues with invariant spacing, which are found in beta-defensins, but the overall amino acid similarity of Tdl to other members of the beta-defensin family is low. In addition, the Tdl gene shows genomic organization similar to that of other beta-defensin genes. We have found that Tdl is specifically expressed in Sertoli cell-lineage in seminiferous cords in embryonic testes, but not in embryonic ovaries after 12.5dpc when the sexual differentiation of gonads is initiated. Tdl is specifically expressed in gonads among adult tissues, and its expression persisted in Sertoli cells.     

Saltatory forward movement of a poly(A) polymerase during poly(A) tail addition

Vaccinia poly(A) polymerase (VP55) interacts with > or = 33-nucleotide (nt) primers via uridylates at two sites (-27/-26 and -10). It adds approximately 30-nt poly(A) tails with a rapid, processive burst in which the first few nt are added without substantial primer movement, and addition of the remaining adenylates is dependent upon a six-uridylate tract at the extreme 3' end of the primer and accompanied by polymerase translocation. Interaction of VP55 with 2-aminopurine (2-AP)-containing primers was associated with a 3-fold enhancement in 2-AP fluorescence. In stopped-flow experiments, fluorescence intensity changed with time during the polyadenylation burst in a manner dependent upon the position of 2-AP, indicating a non-uniform isomerization of the polymerase-primer complex with time consistent with a discontinuous (saltatory) translocation mechanism. Three distinct translocatory phases could be discerned: a -10(U)-binding site forward movement, a -27/-26(UU)-binding site jump to -10, then a -27/-26(UU)-binding site movement further downstream. Poly(A) tail elongation showed no apparent pauses during these isomerizations. Fluorescence changes during polyadenylation of 2-AP-containing primers with short preformed oligo(A) tails reinforced the above observations. Primers composed entirely of oligo(U) (apart from the 2-AP sensor), in which the polymerase modules might be most able to "slide" uniformly, also showed the characteristic saltatory pattern of translocation. These data indicate, for the first time, a discontinuous mode of translocation for a non-templated polymerase.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

Constitutive and regulated expression of the mouse Dinb (Polkappa) gene encoding DNA polymerase kappa

A recently discovered group of novel polymerases are characterized by significantly reduced fidelity of DNA synthesis in vitro. This feature is consistent with the relaxed fidelity required for the replicative bypass of various types of base damage that frequently block high fidelity replicative polymerases. The present studies demonstrate that the specialized DNA polymerase kappa (polkappa) is uniquely and preferentially expressed in the adrenal cortex and testis of the mouse, as well as in a variety of other tissues. The adrenal cortex is the sole site of detectable expression of the Polkappa gene in mouse embryos. This adrenal expression pattern is consistent with a requirement for polkappa for the replicative bypass of DNA base damage generated during steroid biosynthesis. The expression pattern of polkappa in the testis is specific for particular stages of spermatogenesis and is distinct from the expression pattern of several other low fidelity DNA polymerases that are also expressed during spermatogenesis. The mouse (but not the human) Polkappa gene is primarily regulated by the p53 gene and is upregulated in response to exposure to various DNA-damaging agents in a p53-dependent manner.     

A poly(U) polymerase in tobacco leaves.

A poly(U) polymerizing enzyme has been found in healthy and tobacco mosaic virus-infected tobacco leaves and has been partially purified by affinity chromatography on a gel prepared from agarose with chemically coupled RNA. The enzyme is stimulated by Mn-2+ and dependent on a polynucleotide, preferentially poly(A). The synthesis proceeds optimally at pH 7.6 and 25 degrees C. The enzyme is highly specific for UTP and is inhibited by other ribonucleoside triphosphates. The product was partly sensitive to pancreatic ribonuclease. The synthetic reaction is inhibited in the presence of pyrophosphate but insensitive to 10 mM orthophosphate and high levels of cordycepin, rifampicin and actinomycin D. A molecular weight of about 40,000 has been estimated by sucrose gradient analysis and partition cell ultracentrifugation.