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.     

The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene

Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.     

A second ferritin L subunit is encoded by an intronless gene in the mouse

Multiple homologous sequences for the ferritin L subunit are present in mammalian genomes, but so far, only one expressed gene has been described. Here we report the isolation of a cDNA from a mouse bone marrow library, corresponding to an isoform of the mouse ferritin L subunit. This new subunit, that we named Lg, differs from the L subunit of ten amino acids. Specific amplification of mouse genomic DNA using the polymerase chain reaction (PCR) confirmed the presence of this Lg sequence in the mouse genome but also suggested that it must be encoded by an intronless gene. Using a series of different Lg-specific oligonucleotides as probes, we subsequently isolated a genomic clone containing an uninterrupted sequence, identical to the Lg cDNA. This Lg gene lacks introns and does not contain the 28 base pairs (bp) conserved motif usually present at the 5 end of most ferritin mRNAs, which confers translational regulation by iron. When transiently transfected into K562 cells, this Lg genomic clone is actively transcribed, suggesting that, although it possesses the characteristics of a processed pseudogene, it is likely to correspond to the gene encoding this new ferritin subunit.     

The Drosophila glutathione S-transferase 1-1 is encoded by an intronless gene at 87B

The Drosophila glutathione S-transferase 1-1 is a dimer of a 209 amino acid subunit, designated DmGST1. DmGST1 is encoded by a member of a multigene family. Sequence analysis of a genomic clone for GST1 revealed that it is encoded by an intronless gene. We designate this gene and its other family members the GST D genes in the glutathione S-transferase gene superfamily. The Drosophila GST D genes are mapped by in situ hybridization to chromosome 3R at 87B of the polytene chromosome, which is flanked by the two clusters of hsp70 genes at 87A7 and 87C1. Cytogenetic data in the literature indicated that a puff occurred in this region under heat shock. We report that the glutathione S-transferase activity in Kco cells as determined by conjugation with 1-chloro-2,4-dinitrobenzene is elevated slightly to two-fold under heat shock. The implication of this finding is discussed.     

Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene.

Five non-allelic genes which encode five types of alcohol dehydrogenase subunits have been identified in humans. An additional gene (ADH6) and cDNA, whose coding sequences were not highly analogous to any of the known alcohol dehydrogenase subunits, were recently cloned (Yasunami et al., Proc. Natl. Acad. Sci. USA 88, 7610-7614, 1991). The full-length ADH6 cDNA was expressed in the E.coli expression system and in the in vitro translation system of rabbit reticulocytes. The protein produced had its isoelectric point at pH 8.6, optimum pH at pH 10, and a lower Km for benzylalcohol than for ethanol and propanol. These characteristics are compatible to the properties of mu- or sigma-alcohol dehydrogenase isozyme existing in human stomach, indicating that ADH6 gene encodes the mu- or sigma-alcohol dehydrogenase subunit.     

Yeast ribosomal protein S33 is encoded by an unsplit gene.

The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis. The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD. Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed. By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.     

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.     

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.     

The association of a newly discovered protein, called chondrocalcin, with cartilage calcification

A newly identified calcium binding protein called chondrocalcin with two subunits of molecular weight approximately 35 000 has been studied in bovine, rat and human cartilage matrix using a monospecific polyclonal antibody. Although it is present in small amounts in non-calcifying cartilage, it occurs in local high concentrations wherever cartilage calcification is observed, namely in the calcifying part of the growth plate and in calcified articular cartilage. Immunoelectron microscopy revealed that it is present in exactly the same discrete sites where mineral is first detected. Thus it may act as a nucleating agent for apatite formation. It is deposited in the same sites where unusual local high concentrations of proteoglycan and link protein are detected by immunoelectron microscopy. Chondrocalcin may bind either directly or indirectly to these molecules. Its occurrence within hypertrophic chondrocytes immediately prior to its extracellular appearance suggests that it is synthesised and released by these cells. Its absence from osteoid during intramembranous calcification indicates a selective involvement in endochondral calcification.     

The puzzle posed by COMMD1, a newly discovered protein binding Cu(II)

Copper is critically important for cellular metabolism. It plays essential roles in developmental processes, including angiogenesis. The liver is central to mammalian copper homeostasis: biliary excretion is the major route of excretion for ingested copper and serves to regulate the total amount of copper in the organism. An extensive network of proteins manipulates copper disposition in hepatocytes, but comparatively little is known about this protein system. Copper exists in two oxidation states: most extracellular copper is Cu(II) and most, if not all, intracellular copper is Cu(I). Typical intracellular copper-binding proteins, such as the Cu-transporting P-type ATPases ATP7B (Wilson ATPase) and ATP7A (Menkes ATPase), bind copper as Cu(I). Accordingly, the recent discovery that the ubiquitous protein COMMD1 binds Cu(II) exclusively raises the question as to what role Cu(II) may play in intracellular processes. This issue is particularly important in the liver and brain. In humans, Wilson’s disease, due to mutations in ATP7B, exhibits progressive liver damage from copper accumulation; in some Bedlington terriers, mutations in COMMD1 are associated with chronic copper-overloaded liver disease, clinically distinct from Wilson’s disease. It seems unlikely that Cu(II), which generates reactive oxygen species through the Fenton reaction, has a physiological role intracellularly; however, Cu(II) might be the preferred state of copper for elimination from the cell, such as by biliary excretion. We argue that COMMD1 participates in the normal disposition of copper within the hepatocyte and we speculate about that role. COMMD1 may contribute to the mechanism of biliary excretion of copper by virtue of binding Cu(II). Additionally, or alternatively, COMMD1 may be an important component of an intracellular system for utilizing Cu(II), or for detecting and detoxifying it.     

Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.     

The lit gene product which blocks bacteriophage T4 late gene expression is a membrane protein encoded by a cryptic DNA element, e14.

Escherichia coli lit(Con) mutations cause a severe inhibition of gene expression late in infection by bacteriophage T4 owing to the overproduction of one, and possibly two, proteins (C. Kao, E. Gumbs, and L. Snyder, J. Bacteriol. 169:1232-1238, 1987). One or both of these proteins interact, either directly or indirectly, with a short sequence about one-quarter of the way into the major capsid protein gene of T4, and the inhibition occurs when this late gene of the virus is expressed. In this report we show that lit(Con) mutations are up-promoter mutations in the cryptic DNA element e14 and that only one of the proteins, gplit, of about 34 kilodaltons, is required for the inhibition. We have sequenced the lit gene and the surrounding regions. From the sequence, and from cell fractionation studies, we conclude that gplit is an inner membrane protein. Since the assembly of T4 heads is thought to occur on the inner face of the inner membrane, we propose that gplit interferes with a normal regulation which coordinates the synthesis of proteins and the assembly of T4 heads.     

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.     

Some properties of peanut clump, a newly discovered virus

A mechanically transmissible soil-borne virus causing peanut clump disease in Upper Volta is described. It infected mainly species of Chenopodia-ceae and was propagated in Chenopodium amaranticolor. Infectivity was lost from sap of C. amaranticolor after 10 min at 64 °C, and after dilution to 10^-5 but not io^-4. A purification procedure is described. The particles are rod-shaped and of two predominant lengths, 190 and 245 nm. The virus is not serologically related to tobacco rattle, pea early-browning, or soil-borne wheat mosaic viruses, or to a virus associated with a rhizomania-like disease of beet.     

Testis-specific expression of an intronless gene encoding a human poly(A) polymerase.

A mouse intronless gene, encoding a testis-specific poly(A) polymerase (mPAPT), was previously identified. mPAPT may play a role as a putative enzyme that is responsible for polyadenylation regulation during mouse spermatogenesis. In order to understand how PAPT genes are conserved in mammals, we isolated a human cDNA homolog encoding a human PAPT (hPAPT), which was specifically expressed in the testis. The structure of hPAPT was very similar to that of mPAPT. The about 100 residues at the C-terminal region of a nuclear poly(A) polymerase, PAP II, were missing in both PAPT proteins. An analysis of the genomic DNA showed that the hPAPT gene is an intronless gene that is similar to the mPAPT gene. Interestingly, the sequence homology between hPAPT and mPAPT was much lower than the homology between hPAP II and mPAP II. The phylogenetic analysis suggests that PAPTs arose through retrotransposition after the amphibian-amniote split during evolution.     

Degradation of polycyclic aromatic hydrocarbons by a newly discovered enteric bacterium, Leclercia adecarboxylata

A bacterial strain, PS4040, capable of degrading polycyclic aromatic hydrocarbons for use as the sole carbon source was isolated from oily-sludge-contaminated soil. The 16S rRNA gene showed 98.8% homology to that of Leclercia adecarboxylata. Comparative molecular typing with the clinical strain of L. adecarboxylata revealed that there were few comigrating and few distinct amplimers among them.