Characterization of catechol 2,3-dioxygenases.

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.     

Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components

Linear double-stranded (ds)DNA, obtained by excision of the cloned A and B components of tomato golden mosaic virus (TGMV) from recombinant plasmids, was found to infect plants and to elicit symptoms identical to those obtained with TGMV or TGMV DNA. Progeny virus isolated from plants infected with cloned DNA was infective and indistinguishable from TGMV on the basis of (a) its circular single-stranded (ss)DNA genome, (b) its capsid polypeptide, (c) its particle morphology and (d) serological identity. Southern blot analysis of DNA extracted from cells infected with cloned DNA, or TGMV DNA, revealed the same intracellular ss and dsDNA species, represented in both A and B components, except for a subgenomic, possibly defective, DNA, which was not detected in infections with cloned DNA. Infection with cloned DNA was achieved when cloned A and B components were both present, but not with either cloned A or B components separately. TGMV is the first DNA virus for which unequivocal proof of a bipartite genome has been obtained.     

细胞核重新编程的研究进展

细胞核重新编程是哺乳动物正常胚胎和克隆胚胎发育的关键性因素,主要表现为表观遗传学上变化。在受精卵形成和发育过程中,基因组的甲基化状态和组蛋白的结合形式均发生改变;在核移植产生的克隆胚胎中,供体细胞核也会经历核膜破裂、早熟染色体凝集等变化,重新获得分化的潜能而发育为正常的克隆动物。同时存在多种因素影响重新编程的进行。现对哺乳动物细胞核重新编程的研究进展进行综述,以期为该领域进一步的探索提供借鉴。     

A brave new beef: The US food and drug administration's review of the safety of cloned animal products

To meet its public mandate, the US Food and Drug Administration (FDA) collected studies on the potential health hazards of eating or drinking cloned food products. Based on an earlier National Academy of Sciences study that, on closer analysis, was not nearly as sanguine, the FDA's report found no evidence of a health risk from the public's ingestion of cloned food products. This article analyzes the risks the FDA considered, and concludes that there is a disconnect between the risks the FDA assessed in these studies and the risks that might arise from cloned food products. The FDA should consider instituting effective tracking mechanisms and other diagnostics that would permit scientists and the public to answer the question of health risks posed by cloned food products.     

Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos

Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.     

EGF and TGF-alpha supplementation enhances development of cloned mouse embryos

In this study, we sought to determine the extent to which mitogenic growth factors affect the survival and development of cloned mouse embryos in vitro. Cloned embryos derived by intracytoplasmic nuclear injection (ICNI) of cumulus cell nuclei into enucleated oocytes were incubated in culture media supplemented with EGF and/or TGF-alpha for 4 days. Compared to control, treatment with either growth factor significantly increased the blastocyst formation rate, the total number of cells per blastocyst, the cell ratio of the inner cell mass and the trophectoderm (ICM:TE ratio), and EGF-R protein expression in cloned embryos. In most instances these effects were enhanced in cloned embryos when EGF and TGF-alpha were combined. Although fewer blastocysts developed from cloned than from fertilized one-cell stage embryos, growth factor treatment appeared to have the greatest effect on cloned embryos. These results demonstrate that mitogenic growth factors significantly enhance survival and promote the preimplantation development of cloned mouse embryos.     

Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank

Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.     

A procedure for rapid isolation of cloned cDNA inserts using the polymerase chain reaction technique

A reproducible and rapid procedure for isolation of cloned cDNA insert from a lambda gt11 cDNA library is described. The procedure relies on the polymerase chain reaction method using forward and reverse primers for lambda gt11, followed by isolation of the cloned cDNA insert by a rapid technique. The procedure should also be applicable to isolation of cDNA inserts cloned in other vectors such as lambda gt10.     

Production of second-generation cloned cats by somatic cell nuclear transfer

We successfully produced second-generation cloned cats by somatic cell nuclear transfer (SCNT) using skin cells from a cloned cat. Skin cells from an odd-eyed, all-white male cat (G0 donor cat) were used to generate a cloned cat (G1 cloned cat). At 6 months of age, skin cells from the G1 cloned cat were used for SCNT to produce second-generation cloned cats. We compared the in vitro and in vivo development of SCNT embryos that were derived from the G0 donor and G1 cloned donor cat's skin fibroblasts. The nuclei from the G0 donor and G1 cloned donor cat's skin fibroblasts fused with enucleated oocytes with equal rates of fusion (60.7% vs. 58.8%, respectively) and cleavage (66.3% vs. 63.4%). The 2-4-cell SCNT embryos were then transferred into recipients. One of the five recipients of G0 donor derived NT embryos (20%) delivered one live male cloned kitten, whereas 4 of 15 recipients of the G1 cloned donor cat derived NT embryos (26%) delivered a total of seven male second-generation cloned kittens (four live kittens from one surrogate, plus two stillborn kittens, and one live kitten that died 2d after birth from three other surrogate mothers). The four second-generation cloned kittens from the same surrogate all had a white coat color; three of the four second-generation cloned kittens had two blue eyes, and one of the second-generation cloned kittens had an odd-eye color. Despite low cloning efficiency, cloned cats can be used as donor cats to produce second-generation cloned cats.     

Cloning of a galacturonic acid uptake gene from Erwinia chrysanthemi EC16

Abstract A 3.4 kb fragment of Erwinia chrysanthemi EC16 DNA capable of complementing galacturonic acid uptake mutants ( exuT ) was identified and cloned into a multicopy vector. In E. chrysanthemi B374 exuT mutants, the cloned DNA provided for growth of the mutant strains on galacturonic acid by complementing the galacturonic acid uptake defect. Alkaline phosphatase ( phoA ) gene fusions with the cloned DNA suggested that most of the cloned DNA was necessary for complementation of exuT mutant strains. Using anti-alkaline phosphatase antibody, a hybrid ExuT-PhoA protein was localized to the membrane fraction of the bacterium.     

Dairy cattle exploratory and social behaviors: is there an effect of cloning?

While an increasing number of animals are produced by means of somatic cloning, behavioral studies on cloned animals are still rare. The aim of this study was to investigate whether the somatic cloning procedure has an influence on locomotion, exploratory, vocal and social behaviors of heifers. Ten heifers were used in the present study. Five of them were cloned heifers derived from somatic cells of three different Prim'Holstein cows and five others were same-age control heifers produced by artificial insemination. In addition to observations of social behaviors in the stable group, each animal was placed individually for a short time in an unfamiliar environment. Our results failed to show any statistical differences between clones and their controls both in frequencies of agonistic and non-agonistic behaviors. However, cloned heifers showed significantly more non-agonistic and less agonistic behaviors towards other cloned partners than towards control ones. This result also stood for control heifers. As far as their Hierarchical Index was concerned, three cloned heifers were highest ranking and two others lowest ranking. In this herd, social dominance appeared to be linked to body weight and age rather than to a cloning effect. In an unfamiliar environment, cloned and control subjects exhibited the same level of locomotion and vocalization. However, cloned heifers showed more exploratory behaviors than did control ones. This difference could be due to environmental factors during the postnatal period rather than to cloning.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows

Cloning technologies, including embryo splitting and nuclear transfer, were introduced into dairy cattle breeding in the early 1980s. With the recent worldwide attention on the cloning of sheep ("Dolly") and cows ("Gene"), the potential food safety concerns for food products derived from cloned animals needs to be addressed. There has been no study of the composition of milk produced by cloned cows. In this preliminary study, we evaluated the composition of milk from 15 lactating non-embryonic cell cloned cows and six non-cloned lactating cows over a single season. The cloned cows came from five unique genetic lines and three distinct breeds. Milk samples were analyzed for total solids, fat, fatty acid profile, lactose, protein and compared to non-cloned and literature values. Gross chemical composition of milk from cloned cows was similar to that of the non-cloned cows and literature values. Our results lead us to conclude that there are no obvious differences in milk composition produced from cloned cows compared to non-cloned cows.     

Analysis of Bacillus megaterium lipolytic system and cloning of LipA,a novel subfamily I.4 bacterial lipase

The lipolytic system of Bacillus megaterium 370 was investigated, showing the existence of at least two secreted lipases and a cell-bound esterase. A gene coding for an extracellular lipase was isolated and cloned in Escherichia coli. The cloned enzyme displayed high activity on short to medium chain length (C(4)-C(8)) substrates, and poor activity on C(18) substrates. On the basis of amino acid sequence homology, the cloned lipase was classified into subfamily I.4 of bacterial lipases.     

Evaluation of meat products from cloned cattle: biological and biochemical properties

Agricultural utilization of cloned livestock produced by nuclear transfer and their products for food will require public and governmental acceptance. A series of studies of properties of meat derived from cloned cattle was carried out to collect data for the safety assessment of cloned cattle products. Meat samples obtained from embryonic cloned, somatic cloned and non-cloned cattle were analyzed for chemical composition, as well as amino acids and fatty acids. Digestibility, allergenicity, and mutagenicity of meat were also examined. There were no significant differences in these properties among embryonic cloned, somatic cloned and non-cloned cattle. The analyses and tests revealed that there were no significant biological differences in meat from a non-cloned, an embryonic cloned, or a somatic cloned animal. A 14-week feeding trial in rats showed there were no abnormalities in body growth, general condition, locomotor activity, reflexes, sexual cycle, urinalysis, hematology, blood biochemistry, and histology. This study showed for the first time that the biological/biochemical properties of meat of cloned cattle are similar to those of non-cloned cattle.     

Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus)

For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our results suggest similar cognitive capacities of kin and non kin discrimination in AI and cloned animals. Kinship may be a common factor in determining the social grouping within a herd.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Risk assessment of meat and milk from cloned animals

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.