Internal Restriction Sites Research Articles

A programmable DNA roadblock system using dCas9 and multivalent target sites.

A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.

A ligation and restriction enzyme independent cloning technique: an alternative to conventional methods for cloning hard-to-clone gene segments in the influenza reverse genetics system

BackgroundReverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest.MethodsHerein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation.ResultsHard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued.ConclusionThe LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method.

A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477-7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629-8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5'end without changing the amino acid sequence. The syn-pds-int gene (1872bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.

New strategies for the expression of human P‐glycoprotein (MDR1) in the yeast Pichia pastoris

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. The search for P‐gp inhibitors as co‐therapeutics to combat MDR has had little success after decades of studies. We previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. Effects on P‐gp ATPase hydrolysis activity was used as an initial biochemical screen for inhibitor effectiveness followed by assays of MDR‐reversal of a prostate cancer cell line ((Brewer et al. in Mol Pharmacol. 2014; Follit et al in Pharmacol Res Perspect. 2015). More recently, computationally identified compounds were shown to reverse MDR in cancer cells, but did not appear to inhibit ATP hydrolysis by isolated P‐gp. One reason may be that mouse P‐gp rather than human P‐gp was previously used in our biochemical tests. Expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported, but unfortunately, we experienced very modest expression levels of the human P‐gp expression construct for P. pastoris available in our lab. Codon optimization of mouse P‐gp was previously shown by others to result in increased protein yield, therefore a gene for human P‐gp was designed with optimized codon usage for translation in P. pastoris. A strong Kozak sequence was incorporated at the 5′ end of the coding frame. Unique internal restriction sites were incorporated that allow subcloning of individual sections of the gene, if necessary for troubleshooting and future mutagenesis. The DNA was synthesized using standard Gibson assembly methods and cloned into a pUC57 cloning vector. When transformed into E. coli, DNA rearrangement was observed. To identify whether a specific section of the insert was the cause of counter‐selection in E. coli, the insert DNA was digested into 5 component pieces and each one was subcloned. One of the sections was found to be more susceptible to rearrangements. We report here our attempts at constructing a stable, codon‐optimized, human P‐gp gene for high yield protein production in Pichia pastoris.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.

PaperClip: rapid multi-part DNA assembly from existing libraries.

Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.

Expression and purification of SenX3 from the Mycobacterium tuberculosis strain H37Rv in Escherichia coli

Infection with the bacterium Mycobacterium tuberculosis (MTB) causes tuberculosis, a pulmonary infection that may be fatal if left untreated. Misuse or mismanagement of tuberculosis drugs may lead to drug-resistant pathogen forms that are difficult to treat and contribute to a global health problem. The MTB SenX3/RegX3 signal transduction system allows bacteria to externally sense the environment and mediate an appropriate internal response; SenX3 is also associated with MTB virulence, suggesting that this protein may provide a potential therapeutic target. To investigate the role of SenX3 and MTB drug resistance, SenX3 was cloned, expressed and purified in Escherichia coli. SenX3 was cloned from the genome of the MTB strain H37Rv by polymerase chain reaction and an internal NcoI restriction site was destroyed by site-directed mutagenesis to allow cloning into the pET-28b prokaryotic expression vector. SenX3 expression from the resulting pET‑28b-mSenX3 plasmid was induced with isopropyl β-D-thiogalactoside and the protein was purified using Ni-NTA agarose affinity chromatography. A pure protein of the expected size was identified. The examination of purified SenX3 protein is considered to enable the in‑depth investigation of SenX3-mediated drug resistance.

A simple and fast system for cloning influenza A virus gene segments into pHW2000- and pCAGGS-based vectors

The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.

Specific Targeting of the Metallophosphoesterase YkuE to the Bacillus Cell Wall Requires the Twin-arginine Translocation System

The twin-arginine translocation (Tat) pathway is dedicated to the transport of fully folded proteins across the cytoplasmic membranes of many bacteria and the chloroplast thylakoidal membrane. Accordingly, Tat-dependently translocated proteins are known to be delivered to the periplasm of Gram-negative bacteria, the growth medium of Gram-positive bacteria, and the thylakoid lumen. Here, we present the first example of a protein, YkuE of Bacillus subtilis, that is specifically targeted by the Tat pathway to the cell wall of a Gram-positive bacterium. The cell wall binding of YkuE is facilitated by electrostatic interactions. Interestingly, under particular conditions, YkuE can also be targeted to the cell wall in a Tat-independent manner. The biological function of YkuE was so far unknown. Our present studies show that YkuE is a metal-dependent phosphoesterase that preferentially binds manganese and zinc.

Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells

BackgroundCloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert.ResultsUtilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358.ConclusionsOur results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe(2+) > Co(2+) > Zn(2+) > Mn(2+) and Ni(2+). Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe(2+) under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe(2+) and Zn(2+) under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe(2+)-MshB and Zn(2+)-MshB.

Twin PCRs: a simple and efficient method for directional cloning of PCR products

A simple and efficient method was developed for directional cloning of PCR products without any restriction enzyme digestion of the amplified sequence. Two pairs of primers were designed in which parts of two restriction enzyme recognition sequences were integrated, and the primers were used for two parallel PCRs. The PCR products were mixed, heat denatured and re-annealed to generate hybridized DNA fragments bearing sticky ends compatible with restriction enzymes. This method is particularly useful when it is necessary to use a restriction enzyme but there is an additional internal restriction site within the amplified sequence, or when there are problems caused by end sensitivity of restriction enzymes.

Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Summary Aspartic proteases are a relatively small group o f enzymes which express in various nematodes including Onchocerca volvulus . An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic pro tease of O. volvulus , was made by slot blot analysis in a closely relat ed species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that 32 P-labeled cDNA insert. To prepare the initial probe , OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridizatio n of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the 32 P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the sa me probe revealed a single band except when predict ed internal restriction sites were affected. It was co nfirmed that Onchocerca contains a single copy of t he gene corresponding to this cDNA insert per haploid genome.

Resting State Orai1 Diffuses as Homotetramer in the Plasma Membrane of Live Mammalian Cells

Store-operated calcium entry is essential for many signaling processes in nonexcitable cells. The best studied store-operated calcium current is the calcium release-activated calcium (CRAC) current in T-cells and mast cells, with Orai1 representing the essential pore forming subunit. Although it is known that functional CRAC channels in store-depleted cells are composed of four Orai1 subunits, the stoichiometric composition in quiescent cells is still discussed controversially: both a tetrameric and a dimeric stoichiometry of resting state Orai1 have been reported. We obtained here robust and similar FRET values on labeled tandem repeat constructs of Orai1 before and after store depletion, suggesting an unchanged tetrameric stoichiometry. Moreover, we directly visualized the stoichiometry of mobile Orai1 channels in live cells using a new single molecule recording modality that combines single molecule tracking and brightness analysis. By alternating imaging and photobleaching pulses, we recorded trajectories of single, fluorescently labeled Orai1 channels, with each trajectory consisting of bright and dim segments, corresponding to higher and lower numbers of colocalized active GFP label. The according brightness values were used for global fitting and statistical analysis, yielding a tetrameric subunit composition of mobile Orai1 channels in resting cells.

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the "T-vector" can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3'-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5'-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.

Identification of a 35-Kilodalton Mycobacterium tuberculosis Protein Containing B- and T-Cell Epitopes

Abstract Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

Internal Restriction Sites: Quality Assurance Aids in Genotyping

Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or incorporate a second native site within the PCR amplicon, provide the mechanism by which efficiency of restriction enzymes can be assessed with every sample. The outcome is confirmation of the activity of the discriminating enzyme regardless of genotype.

Humanized β-Thalassemia Mouse Model Containing the Common IVSI-110 Splicing Mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.

Primer Prim'er: a web based server for automated primer design.

The Northeast Structural Genomics Consortium (NESG) is one of nine NIH-funded pilot projects created to develop technologies needed for structural studies of proteins on a genome-wide scale. One of the most challenging aspects of this emerging field is the production of protein samples amenable to structural determination. To do this efficiently, all steps in the protein production pipeline must be automated. Here we describe the Primer program (linked from http://www-nmr.cabm.rutgers.edu/bioinformatics, www-nmr.cabm.rutgers.edu/bioinformatics, a web-based primer design program freely available to the scientific community, which was created to automate this time consuming and laborious task. This program has the ability to simultaneously calculate plasmid specific primer sets for multiple open reading frame (ORF) targets, including 96-well and greater formats. Primer includes a library of commonly used plasmid systems and possesses the ability to upload user-defined plasmid systems. In addition to calculating gene-specific annealing regions for each target, the program also adds appropriate restriction endonuclease recognition or viral recombination sites while preserving a reading frame with plasmid based fusions. Primer has several useful features such as sorting calculated primer sets by target size, facilitating interpretation of PCR amplifications by agarose gel electrophoresis, as well as supplying the molecular biologist with many important characteristics of each target such as the expected size of the PCR amplified DNA fragment and internal restriction sites. The NESG has cloned over 1500 genes using oligonucleotide primers designed by Primer.

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens.

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences. The tandem repeats are 229-246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin. Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.