In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication

1979 ◽  
Vol 169 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Volker Nüsslein-Crystalla ◽  
Ursula Scheefers-Borchel
Keyword(s):  
Dna Replication ◽  
E Coli ◽  
In Vitro Replication ◽  
Dna Fragment

Analysis of transcriptional regulation of the s38 chorion gene of Drosophila by P element-mediated transformation

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 137-146
Author(s):  
Laura Kalfayan ◽  
Barbara Wakimoto ◽  
Allan Spradling
Keyword(s):  
Transcriptional Regulation ◽  
P Element ◽  
Developmental Expression ◽  
Developmental Profile ◽  
E Coli ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
P Element Transformation ◽  
Lac Z

Transcriptional regulation of the s38 chorion gene was studied using P element-mediated germline transformation. A 5·27 kb DNA fragment containing the s38 gene and 5′- and 3′-flanking sequences, was tested for its ability to be transcribed with correct developmental specificity. Five single-insert transformed lines were generated by microinjection of this DNA fragment cloned into a marked P element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native s38 gene. The 1·3 kb at the 5′ end of this tested fragment was fused to the E. coli lac z gene. This fragment was also capable of initiating transcription of E. coli lac z RNA with the developmental profile of the native s38 gene. In vitro deletion studies are underway to determine which sequences in the 1·3 kb fragment are necessary for regulating the developmental expression of the gene.

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals Gene Cluster Responsible for Validamycin Biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

2005 ◽  
Vol 71 (9) ◽  
pp. 5066-5076 ◽  
Author(s):  
Yi Yu ◽  
Linquan Bai ◽  
Kazuyuki Minagawa ◽  
Xiaohong Jian ◽  
Lei Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Control Agent ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
E Coli ◽  
Sheath Blight Disease ◽  
Dna Fragment ◽  
Blight Disease

ABSTRACT A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

scholarly journals A Fifteen-Amino-Acid Peptide Inhibits Human Papillomavirus E1-E2 Interaction and Human Papillomavirus DNA Replication In Vitro

1998 ◽  
Vol 72 (10) ◽  
pp. 8166-8173 ◽  
Author(s):  
Hiroaki Kasukawa ◽  
Peter M. Howley ◽  
John D. Benson
Keyword(s):  
Human Papillomavirus ◽  
Amino Acid ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Hpv 16 ◽  
Hpv Dna ◽  
Amino Acid Peptide ◽  
In Vitro Replication ◽  
Papillomavirus Dna

ABSTRACT Mutation of the conserved glutamic acid residue at position 39 of human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its E1 interaction activity and its replication function in transient replication assays but does not affect E2 transcriptional activation. This E39A mutation also disrupts replication activity of HPV-16 E2 in HPV-16 in vitro DNA replication. On this basis, we designed 23- and 15-amino-acid peptides derived from HPV-16 E2 sequences flanking the E39 residue and tested the ability of these peptides to inhibit interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity of these peptides was specific, since analogous peptides in which alanine was substituted for the E39 residue did not inhibit interaction. The 15-amino-acid peptide E2N-WP15 was the smallest peptide tested that effectively inhibited HPV-16 E1-E2 interaction. This peptide also inhibited in vitro replication of HPV-16 DNA. The efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11 and HPV-16 and inhibited in vitro replication with these same combinations of E1 and E2 proteins. These results provide further evidence that E1-E2 interaction is required for papillomavirus DNA replication and constitute the first demonstration that inhibition of this interaction is sufficient to prevent HPV DNA replication in vitro.

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication

1994 ◽  
Vol 14 (6) ◽  
pp. 4173-4182
Author(s):  
I Kawasaki ◽  
Y S Bae ◽  
T Eki ◽  
Y Kim ◽  
H Ikeda
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Alpha Satellite ◽  
E Coli ◽  
Replication System ◽  
Satellite Repeats

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Electron-microscopic mapping of AT-rich regions and of E. coli RNA polymerase-binding sites on the circular kinetoplast DNA of Trypanosoma cruzi

1975 ◽  
Vol 17 (3) ◽  
pp. 287-306
Author(s):  
C. Brack ◽  
E. Delain
Keyword(s):  
Dna Replication ◽  
Trypanosoma Cruzi ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Specific Binding ◽  
Kinetoplast Dna ◽  
Electron Microscopic ◽  
E Coli ◽  
Polymerase Binding

Partial alkaline denaturation of the circular kinetoplast DNA (kDNA) of Trypanosoma cruzi has shown the existence of 4 small, well-defined AT-rich regions with an average size of about 200 base pairs. They are almost equally distributed, separated by approximately 90 degrees on the circular molecule. All minicircles, whether free or linked in networks, have the same denaturation pattern and, therefore, seem to contain the same information. The long linear molecules present in low amounts in the kDNA samples do not show the same denaturation pattern. Partial denaturation of molecules in larger associations indicates that the circular units may be linked to each other by one strand only. kDNA can be transcribed in vitro by the RNA polymerase of E. coli. RNA polymerase-kDNA complexes have been studied in the electron microscope. By spreading the DNA-protein complexes by adhesion to positively charged carbon films and dark-field observation, it was possible to show the existence of 4 specific binding sites of the E. coli RNA polymerase on the kDNA circles. Comparing the position of the polymerase-binding sites and the AT-rich melted zones, it is suggested that a correlation exists between the two. As had been shown in earlier work, the replication of circular kDNA can be blocked by treating the trypanosomes with the trypanocidal drug Berenil. The comparison of the relative position of the Berenil-blocked replication forks with the position of the 4 denaturation loops shows that the DNA replication is stopped at these AT-rich regions. Since there is evidence that Berenil binds preferentially to AT-rich DNA and seems to be involved in inhibition of DNA replication, the following hypothetical model can be proposed. The replication of the circular kDNA molecules is discontinuous and involves the synthesis of RNA primers; when Berenil is bound to the AT-rich regions, synthesis of new RNA primers is inhibited and replication is blocked at these points, leading to the accumulation of replicating intermediates with defined branch lengths.

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