A multicopy vector system for genetic studies in<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> and other zygomycetes

Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu^- phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.Communicated by E. Cerdà-OlmedoOn 1 January, 2004, the Biotechnological Institute became Bioneer A/S ()     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M.?circinelloides OPRTase with those of E. coli and S.?typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.     

人促红细胞生成素基因组基因的克隆及全序列分析

为了克隆到全长的人促红细胞生成素基因,从而在真核系统中及转基因动物乳腺中进行表达,从一个中国人的血细胞中提取基因组ＤＮＡ,构建了不完全基因组噬菌体文库,文库效价为５×１０４．这种文库的构建方法是用ＢａｍＨⅠ对基因组ＤＮＡ进行完全酶切,回收１０．５ｋｂ左右的酶切片段,插入到λ－噬菌体ＥＭＢＬ３载体中．筛选文库所使用的探针是用ＰＣＲ方法从基因组ＤＮＡ模板中扩增的５５６ｂｐ的ＥＰＯ基因片段．从该文库中筛选到了一个含有完整ＥＰＯ基因的插入片段长度为１２．５ｋｂ的阳性克隆．经全序列分析证实,所克隆的ＥＰＯ基因编码正确,但在５′－侧翼及第一内含子处与国外发表的序列相比较,发现两处核苷酸差异,这两个核苷酸的差异改变了５′－调控区的两个小的开放阅读框——ＯＲＦ１和ＯＲＦ３,其在基因表达调探方面的作用值得进一步探讨．     

Conditional gene vectors regulated in cis

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.     

Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5′ or 3′ to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.     

In VitroProtein-primed Initiation of Pneumococcal Phage Cp-1 DNA Replication Occurs at the Third 3′ Nucleotide of the Linear Template: A Stepwise Sliding-back Mechanism

Phage Cp-1 fromStreptococcus pneumoniaemakes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5′ dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5′ end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infectedS. pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication. A mutational analysis of the 3′ terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and thatin vitroinitiation of Cp-1 DNA replication is directed by the third nucleotide of the template. However, the two terminal nucleotides are recovered during the first steps of elongation. A new variant of the sliding-back mechanism for protein-primed initiation, firstly described forBacillus subtilisphage φ29, is proposed to account for the maintenance of Cp-1 DNA ends. The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Defective Interfering Particles of Vesicular Stomatitis Virus: Functions of the Genomic Termini

The functions of thecis-acting sequence elements at the termini of the negative strand RNA virus vesicular stomatitis virus and its defective-interfering particles were evaluated. Either genomic terminus could signal replication but increasing the complementarity of the termini enhanced replication irrespective of whether the termini consisted of the 3′ leader and its complement or the 5′ trailer and its complement. The 5′ trailer region contains an essentialcis-acting requirement for assembly of RNPs into infectious particles. These findings explain why the majority of DI RNAs are of the 5′ copy-back class: RNAs with complementary termini from either end have a replicative advantage, but only 5′ copy-back RNAs contain the signal for assembly into particles.     

Chlamydophila pneumoniae endonuclease IV prefers to remove mismatched 3′ ribonucleotides: Implication in proofreading mismatched 3′-terminal nucleotides in short-patch repair synthesis

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3′–5′ exonuclease, which is required for removing an incorrectly incorporated 3′-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3′–5′ exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3′–5′ exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3′–5′ exonuclease activity that prefers to remove mismatched 3′-terminal nucleotides in the nick, gap, and 3′ recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3′-terminal nucleotide using the dsDNA with a nick or 3′ recess as substrate. Upon proofreading of the mismatched 3′-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.     

Relationship of the [psi] factor with other plasmids of Saccharomyces cerevisiae

The [psi] factor is an extrachromosomally inherited genetic determinant of the yeast Saccharomyces cerevisiae for which no autonomous physical determinant has yet been identified. Using both physical and genetical techniques we demonstrate that the [psi] determinant does not reside on other previously described yeast extrachromosomally inherited determinants; namely mitochondrial DNA, L double-stranded RNA, M double-stranded RNA, and [2 μm] DNA. Stable [psi⁺] strains, lacking one or other of these determinants were constructed. It is concluded that the [psi] factor may represent a new yeast plasmid.     

Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.     

Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.