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Xanthobacter aminoxidans is a Gram-negative pleomorphic and diazotrophic Knallgas bacillus that undergoes asymmetric budding of V-shaped branched cells during cell division. Like other Xanthobacter spp., cells are yellow from production of zeaxanthine dirhamnoside. We sequenced strain 14a T (= ATCC BAA-299 T ) and report a genome size of 5,829,486 bp with a G+C content of 67.9%.

The glycopeptide antibiotic-producing soil actinobacterium Kibdelosporangium philippinense A80407 (=ATCC 49844) was sequenced using Illumina and Nanopore sequencing methodologies, and a hybrid genome assembly was generated for this type strain, with a total predicted genome length of 12,054,556 bp, 10,953 protein-coding sequences, 79 RNAs, 298 pseudogenes, and a G+C content of 65.13%.

The hot springs bacterium Litorilinea aerophila PRI-4131 T (= ATCC BAA-2444 T ) was found in Isafjardardjup, in northwest Iceland. In this paper, we present a draft genome sequence for the type strain, with a total predicted genome length of 6,043,010 bp, 4,608 protein-coding sequences, 54 RNAs, 9 CRISPR arrays, and a G+C content of 64.61%.

Ureibacillus terrenus TH9A T (=ATCC BAA-384 T ) was isolated from uncultivated soil in Italy in 1995. We present a draft genome sequence for the type strain, with a predicted genome length of 2,936,851 bp, containing 2,766 protein-coding genes, 82 RNA genes, and 5 CRISPR arrays, with a G+C content of 42.5%.

We characterized the complete genome sequence of actinobacteriophage Yavru ( Siphoviridae ), a cluster FE bacteriophage infecting Arthrobacter globiformis NRRL B-2979; it was 89.5% identical to cluster FE phage Whytu, with a capsid width of 50 nm and a tail length of 90 nm. The genome was 15,193 bp in length, with 23 predicted protein-coding genes.

Bacillus thuringiensis is an agriculturally significant bacterium and common biological pesticide. B. thuringiensis strain MW was isolated from a freshwater stream in Mont Vernon, NH, and sequenced. A draft genome assembly of 5,935,630 bp with a G+C content of 34.86% and an N 50 value of 1,154,949 bp was generated., (Copyright © 2020 Williams and MacLea.)

Dermacoccus nishinomiyaensis is a common bacterial resident of the human skin microbiome, among other environments. D. nishinomiyaensis strain TSA37 was isolated from the ash pan of a residential wood pellet stove. A genome assembly of 3,130,592 bp was generated, with an N 50 value of 197,547 bp and a calculated G+C content of 69.01%., (Copyright © 2019 Williams and MacLea.)

The pigmented bacterium Deinococcus radiophilus , which is highly resistant to radiation exposure, was first isolated from irradiated lizardfish. We report a genome assembly of D. radiophilus UWO 1055 T (=ATCC 27603 T ), with a predicted genome size of 2.7 Mbp (62.66% G+C content). A number of CRISPR-associated proteins and two CRISPR arrays were identified., (Copyright © 2019 Maynard and MacLea.)

Lentibacillus salicampi SF-20 T (=ATCC BAA-719 T ) was first isolated from a Yellow Sea salt field in Korea in 2002. Here, we report that the L. salicampi ATCC BAA-719 T genome sequence has a predicted length of 3,897,716 bp, containing 3,945 total genes and a CRISPR array, with a G+C content of 43.0%., (Copyright © 2019 Simoes Junior and MacLea.)

Xanthobacter tagetidis is a thiophene-degrading bacterium associated with root balls of the plant genus Tagetes , which includes marigolds. It is a Gram-negative facultatively autotrophic bacterium with pleomorphic morphology exhibiting bent and branching rods. From strain TagT2C T (= ATCC 700314 T ), we report a genome assembly of 4,945,221 bp and a 69.5% G+C content., (Copyright © 2019 Fatima et al.)

The aerobic, Gram-positive, psychrotolerant bacterium Kurthia sibirica was first isolated from the stomach and intestinal contents of the Magadan mammoth recovered from the permafrost in eastern Siberia in 1977. K. sibirica was sequenced, and the predicted genome size is 3,496,665 bp, with 36.42% G+C content.

The Gram-negative genus Kangiella contains a number of halophilic species that display high levels of iso-branched fatty acids. Kangiella spongicola was isolated from a marine sponge, Chondrilla nucula, from the Florida Keys in the United States. A genome assembly of 2,825,399 bp with a 44.31% G+C content was generated from strain A79 T (=ATCC BAA-2076 T ).

The genome of the type strain of the Kurthia genus, Kurthia zopfii ATCC 33403, was sequenced. Nonpathogenic K. zopfii has been isolated from intestinal contents, fecal material, meats, meat products, milk, water, and air, including air at high altitudes. The predicted genome size is 2,878,279 bp, with 37.05% G+C content.

Genomes from three strains of Kocuria rosea were sequenced. K. rosea ATCC 186, the type strain, was 3,958,612 bp in length with a total G+C content of 72.70%. When assembled, K. rosea ATCC 516 was 3,862,128 bp with a 72.82% G+C content. K. rosea ATCC 49321 was 4,018,783 bp in size with a 72.49% G+C content., (Copyright © 2018 Trachtenberg et al.)

Oceanimonas doudoroffii ATCC 27123 T is an obligately aerobic Gram-negative rod of the class Gammaproteobacteria It was first isolated from surface seawater off the coast of Oahu, HI, USA, in 1972. The predicted genome size is 3,832,938 bp (G+C content, 60.03%), which contains 3,524 predicted coding sequences., (Copyright © 2017 Brennan et al.)

The aerobic phenol-degrading Gram-negative rod Oceanimonas baumannii ATCC 700832 T was first isolated from estuary mud from the River Wear, United Kingdom, in 1983. Information on the draft genome sequence for O. baumannii ATCC 700832 T is included in this announcement. The predicted genome size is 3,809,332 bp, with 55.88% G+C content., (Copyright © 2017 McClelland et al.)

Staphylococcus epidermidis ATCC 12228 was sequenced using a long-read method to generate a complete genome sequence, including some plasmid sequences. Some differences from the previously generated short-read sequence of this nonpathogenic and non-biofilm-forming strain were noted. The assembly size was 2,570,371 bp with a total G+C% content of 32.08%., (Copyright © 2017 MacLea and Trachtenberg.)

Oceanospirillum multiglobuliferum ATCC 33336 T is a motile gammaproteobacterium with bipolar tufted flagella, noted for its low salt tolerance compared to other marine spirilla. This strain was originally isolated from the putrid infusions of Crassostrea gigas near Hiroshima, Japan. This paper presents a draft genome sequence for O. multiglobuliferum ATCC 33336 T ., (Copyright © 2017 Carney et al.)

Oceanospirillum linum ATCC 11336 T is an aerobic, bipolar-tufted gammaproteobacterium first isolated in the Long Island Sound in the 1950s. This announcement offers a genome sequence for O. linum ATCC 11336 T , which has a predicted genome size of 3,782,189 bp (49.13% G+C content) containing 3,540 genes and 3,361 coding sequences., (Copyright © 2017 Trachtenberg et al.)

While philosophers in ancient times had many ideas for the cause of contagion, the modern study of infective agents began with Fracastoro's 1546 proposal that invisible "spores" spread infectious disease. However, firm categorization of the pathogens of the natural world would need to await a mature germ theory that would not arise for 300 years. In the 19th century, the earliest pathogens described were bacteria and other cellular microbes. By the close of that century, the work of Ivanovsky and Beijerinck introduced the concept of a virus, an infective particle smaller than any known cell. Extending into the early-mid-20th century there was an explosive growth in pathogenic microbiology, with a cellular or viral cause identified for nearly every transmissible disease. A few occult pathogens remained to be discovered, including the infectious proteins (prions) proposed by Prusiner in 1982. This review discusses the prions identified in mammals, yeasts, and other organisms, focusing on the amyloid-based prions. I discuss the essential biochemical properties of these agents and the application of this knowledge to diseases of protein misfolding and aggregation, as well as the utility of yeast as a model organism to study prion and amyloid proteins that affect human and animal health. Further, I summarize the ideas emerging out of these studies that the prion concept may go beyond proteinaceous infectious particles and that prions may be a subset of proteins having general nucleating or seeding functions involved in noninfectious as well as infectious pathogenic protein aggregation., (© 2017 Elsevier Inc. All rights reserved.)

Molting in decapod crustaceans is controlled by molt-inhibiting hormone (MIH), an eyestalk neuropeptide that suppresses production of ecdysteroids by a pair of molting glands (Y-organs or YOs). Eyestalk ablation (ESA) activates the YOs, which hypertrophy and increase ecdysteroid secretion. At mid premolt, which occurs 7-14days post-ESA, the YO transitions to the committed state; hemolymph ecdysteroid titers increase further and the animal reaches ecdysis ~3weeks post-ESA. Two conserved signaling pathways, mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β), are expressed in the Gecarcinus lateralis YO. Rapamycin, an mTOR antagonist, inhibits YO ecdysteroidogenesis in vitro. In this study, rapamycin lowered hemolymph ecdysteroid titer in ESA G. lateralis in vivo; levels were significantly lower than in control animals at all intervals (1-14days post-ESA). Injection of SB431542, an activin TGF-β receptor antagonist, lowered hemolymph ecdysteroid titers 7 and 14days post-ESA, but had no effect on ecdysteroid titers at 1 and 3days post-ESA. mRNA levels of mTOR signaling genes Gl-mTOR, Gl-Akt, and Gl-S6k were increased by 3days post-ESA; the increases in Gl-mTOR and Gl-Akt mRNA levels were blocked by SB431542. Gl-elongation factor 2 and Gl-Rheb mRNA levels were not affected by ESA, but SB431542 lowered mRNA levels at Days 3 and 7 post-ESA. The mRNA level of an activin TGF-β peptide, Gl-myostatin-like factor (Mstn), increased 5.5-fold from 0 to 3days post-ESA, followed by a 50-fold decrease from 3 to 7days post-ESA. These data suggest that (1) YO activation involves an up regulation of the mTOR signaling pathway; (2) mTOR is required for YO commitment; and (3) a Mstn-like factor mediates the transition of the YO from the activated to the committed state., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Idiomarina zobellii was isolated from the northwest Pacific Ocean at a depth of 4,000 to 5,000 m in 1985. The draft whole-genome shotgun sequence of I. zobellii KMM 231(T) described in this paper has a predicted length of 2,602,160 bp, containing 2,570 total genes, 52 tRNAs, and a G+C content of 47.10%., (Copyright © 2015 Mithoefer et al.)

Idiomarina abyssalis KMM 227(T) is an aerobic flagellar gammaproteobacterium found at a depth of 4,000 to 5,000 m below sea level in the Pacific Ocean. This paper presents a draft genome sequence for I. abyssalis KMM 227(T), with a predicted composition of 2,684,812 bp (47.15% G+C content) and 2,611 genes, of which 2,508 were predicted coding sequences., (Copyright © 2015 Rheaume et al.)

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In decapod crustaceans, regulation of molting is controlled by the X-organ/sinus gland complex in the eyestalks. The complex secretes molt-inhibiting hormone (MIH), which suppresses production of ecdysteroids by the Y-organ (YO). MIH signaling involves nitric oxide and cGMP in the YO, which expresses nitric oxide synthase (NOS) and NO-sensitive guanylyl cyclase (GC-I). Molting can generally be induced by eyestalk ablation (ESA), which removes the primary source of MIH, or by multiple leg autotomy (MLA). In our work on Carcinus maenas, however, ESA has limited effects on hemolymph ecdysteroid titers and animals remain in intermolt at 7 days post-ESA, suggesting that adults are refractory to molt induction techniques. Consequently, the effects of ESA and MLA on molting and YO gene expression in C. maenas green and red color morphotypes were determined at intermediate (16 and 24 days) and long-term (~90 days) intervals. In intermediate-interval experiments, ESA of intermolt animals caused transient twofold to fourfold increases in hemolymph ecdysteroid titers during the first 2 weeks. In intermolt animals, long-term ESA increased hemolymph ecdysteroid titers fourfold to fivefold by 28 days post treatment, but there was no late premolt peak (>400 pg μl(-1)) characteristic of late premolt animals and animals did not molt by 90 days post-ESA. There was no effect of ESA or MLA on the expression of Cm-elongation factor 2 (EF2), Cm-NOS, the beta subunit of GC-I (Cm-GC-Iβ), a membrane receptor GC (Cm-GC-II) and a soluble NO-insensitive GC (Cm-GC-III) in green morphs. Red morphs were affected by prolonged ESA and MLA treatments, as indicated by large decreases in Cm-EF2, Cm-GC-II and Cm-GC-III mRNA levels. ESA accelerated the transition of green morphs to the red phenotype in intermolt animals. ESA delayed molting in premolt green morphs, whereas intact and MLA animals molted by 30 days post treatment. There were significant effects on YO gene expression in intact animals: Cm-GC-Iβ mRNA increased during premolt and Cm-GC-III mRNA decreased during premolt and increased during postmolt. Cm-MIH transcripts were detected in eyestalk ganglia, the brain and the thoracic ganglion from green intermolt animals, suggesing that MIH in the brain and thoracic ganglion prevents molt induction in green ESA animals.

Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Numerous proteins contain domains that are enriched in glutamine and asparagine residues, and aggregation of some of these proteins has been linked to both prion formation in yeast and a number of human diseases. Unfortunately, predicting whether a given glutamine/asparagine-rich protein will aggregate has proven difficult. Here we describe a recently developed algorithm designed to predict the aggregation propensity of glutamine/asparagine-rich proteins. We discuss the basis for the algorithm, its limitations, and usage of recently developed online and downloadable versions of the algorithm.

Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.

The unexpected discovery of two prions, [URE3] and [PSI+], in Saccharomyces cerevisiae led to questions about how many other proteins could undergo similar prion-based structural conversions. However, [URE3] and [PSI+] were discovered by serendipity in genetic screens. Cataloging the full range of prions in yeast or in other organisms will therefore require more systematic search methods. Taking advantage of some of the unique features of prions, various researchers have developed bioinformatic and experimental methods for identifying novel prion proteins. These methods have generated long lists of prion candidates. The systematic testing of some of these prion candidates has led to notable successes; however, even in yeast, where rapid growth rate and ease of genetic manipulation aid in testing for prion activity, such candidate testing is laborious. Development of better methods to winnow the field of prion candidates will greatly aid in the discovery of new prions, both in yeast and in other organisms, and help us to better understand the role of prions in biology.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S₁) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

While rearing birds in confinement and at high density are very successful practices for producing poultry meat and eggs, these conditions may promote the spread of infectious diseases. Consequently, the poultry industry places greatemphasis on disease control measures, primarily at the animal husbandry level. The field of genomics offers great promise to complement these current control measures by providing information on the molecular basis for disease, disease resistance, and vaccinal immunity. This briefly summarizes some of our efforts to apply several genomic and functional genomics approaches to identify genes and pathways that confer genetic resistance to Marek's disease (MD), a herpesvirus-induced T cell lymphoma of chickens. By utilizing the "top-down" approach of QTL to identify genomics regions, and integrating it with "bottom-up" approaches of transcript profiling and Marek's disease virus (MDV)-chicken protein-protein interactions, three genes that confer resistance to MD are revealed, plus a number of other positional candidate genes of high confidence. These genes can be further evaluated in poultry breeding programmes to determine if they confer genetic resistance to MD. This integrative genomics strategy can be applied to other infectious diseases. The impact of the genome sequence and other technological advancements are also discussed.

Acid endonucleases of the deoxyribonuclease II (DNase II, EC 3.1.22.1) family have been implicated in the degradation of DNA from apoptotic cell corpses formed in the process of normal mammalian development. Although a predicted DNase II has been detected in the chicken through expressed sequence tag (EST) analysis, to date no homolog of these important enzymes has been identified in vivo in any avian species. Here we report the cloning and expression of DNase II from the chicken, Gallus gallus. When expressed, the 363 amino acid glycoprotein is observed to be approximately 45 kDa in size and to exhibit DNA hydrolytic activity at pH 5 consistent with DNase II in other species. Furthermore, chicken DNase II sequence is compared with an identified partial sequence from the zebra finch, Taeniopygia guttata, as well as the previously identified homologs found in the fowlpox and canarypox viruses and the previously cloned mammalian DNases II. Through analysis of its amino acid sequence, comparative gene structure, and conserved synteny, chicken DNase II appears to represent a member of the DNase IIbeta subfamily and the apparent lack of a DNase IIalpha homolog in the chicken has important evolutionary implications for the study of this gene family.

DNase II alpha (EC 3.1.22.1) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3'-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure-activity relationship has been largely unexamined. Recently, we demonstrated that active DNase II alpha consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415-421]. The present paper describes further work to examine the elements of DNase II alpha protein required for activity. Truncated forms and site-specific mutants were expressed in DNase II alpha-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase II alpha contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase II alpha that lacks the purported active-site His(295) can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase II alpha protein structure, which is important for three-dimensional structural analysis and for production of DNase II alpha as a potential protein therapeutic for cystic fibrosis or other disorders.

Deoxyribonuclease IIalpha (DNase IIalpha) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted. Though its Caenorhabditis elegans homolog, NUC-1, is required for digesting DNA of apoptotic cell corpses and dietary DNA, it is not required for viability. However, DNase IIalpha is required in mice for correct development and viability, because undigested cell corpses lead to lesions throughout the body. Recently, we showed that, in contrast to previous reports, active DNase IIalpha consists of one contiguous polypeptide. To better analyze DNase II protein structure and determine residues important for activity, extensive database searches were conducted to find distantly related family members. We report 29 new partial or complete homologs from 21 species. Four homologs with differences at the purported active site histidine residue were detected in the parasitic nematodes Trichinella spiralis and Trichinella pseudospiralis. When these mutations were reconstructed in human DNase IIalpha, the expressed proteins were inactive. DNase II homologs were also identified in non-metazoan species. In particular, the slime-mold Dictyostelium, the protozoan Trichomonas vaginalis, and the bacterium Burkholderia pseudomallei all contain sequences with significant similarity and identity to previously cloned DNase II family members. We report an analysis of their sequences and implications for DNase II protein structure and evolution.

Deoxyribonuclease IIalpha (DNase IIalpha) is one of many endonucleases implicated in DNA digestion during apoptosis. We produced mice with targeted disruption of DNase IIalpha and defined its role in apoptosis. Mice deleted for DNase IIalpha die at birth with many tissues exhibiting large DNA-containing bodies that result from engulfed but undigested cell corpses. These DNA-containing bodies are pronounced in the liver where fetal definitive erythropoiesis occurs and extruded nuclei are degraded. They are found between the digits, where apoptosis occurs, and in many other regions of the embryo. Defects in the diaphragm appear to cause death of the mice due to asphyxiation. The DNA in these bodies contains 3'-hydroxyl ends and therefore stain positive in the TUNEL assay. In addition, numerous unengulfed TUNEL-positive cells are observed throughout the embryo. Apoptotic cells are normally cleared rapidly from a tissue; hence the persistence of the DNA-containing bodies and TUNEL-positive cells identifies sites where apoptosis occurs during development. These results demonstrate that DNase IIalpha is not required for the generation of the characteristic DNA fragmentation that occurs during apoptosis but is required for degrading DNA of dying cells and this function is necessary for proper fetal development.

Deoxyribonuclease IIalpha (DNase IIalpha) is an acid endonuclease found in lysosomes, nuclei, and various secretions. Murine DNase IIalpha is required for digesting the DNA of apoptotic cells after phagocytosis and for correct development and viability. DNase IIalpha purified from porcine spleen was previously shown to contain three peptides, two of which were thiol crosslinked, all derived by processing of a single polypeptide. Commercial bovine protein is consistent with this structure. However, screening of 18 human cell lines failed to demonstrate this processing, rather a 45 kDa protein was consistently observed. Incubation of cells with the N-glycosylation inhibitor tunicamycin resulted in a 37 kDa protein, which is close to the predicted formula weight. The protein also contains at least one thiol crosslink. Similar results were obtained with overexpressed DNase IIalpha. These results suggest that active DNase IIalpha consists of one contiguous polypeptide. We suggest the previous structure reflects proteolysis during protein purification., ((c)2002 Elsevier Science (USA).)

Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.