The development of microsatellite DNA markers for genetic analysis in Douglas-fir

2002 ◽  
Vol 32 (11) ◽  
pp. 1904-1915 ◽  
Author(s):  
Vindhya Amarasinghe ◽  
John E Carlson
Keyword(s):  
Pcr Primers ◽  
Douglas Fir ◽  
Base Pairs ◽  
Genomic Libraries ◽  
Polymorphic Loci ◽  
Microsatellite Dna Markers ◽  
Pcr Products ◽  
Conifer Tree ◽  
Flanking Sequences ◽  
Locus Allele

The microsatellite motifs AG, AC, and ATG were found to be the most abundant in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and several other conifer tree species among di-, tri-, and tetra-nucleotide simple sequence repeats (SSR). Colonies containing AG, AC, and ATG repeats were selected from enriched genomic libraries of Douglas-fir, and 603 were sequenced. Polymerase chain reaction (PCR) primers were designed from flanking sequences in 102 of the SSR clones, of which 50 primer pairs (for 10 AC-repeat microsatellites and 40 AG-repeat microsatellites) produced robust amplification products. Variability was confirmed with 24 unrelated Douglas-fir trees and Medelian segregation with 33-66 progeny from 3 full-sib populations. Forty-eight of the 50 loci were polymorphic, with a mean of 7.5 alleles per locus. Allele sizes ranged from 73 to 292 base pairs. Allele frequencies for the 48 polymorphic loci varied from 0.017 to 0.906 with mean allele frequency of 0.250. Expected heterozygosities among the polymorphic loci varied from 0.174 to 0.926, with a mean of 0.673. Additional, high molecular weight PCR products were amplified by some of the primer pairs, but they did not interfere with the scoring of alleles. Most of the Douglas-fir primer pairs also amplified SSR-containing loci in other conifer species.

Microsatellite sequences in a conifer, Pinus sylvestris

Genome ◽  
1995 ◽  
Vol 38 (6) ◽  
pp. 1244-1248 ◽  
Author(s):  
Silja Kostia ◽  
Sirkka-Liisa Varvio ◽  
Pekka Vakkari ◽  
Pertti Pulkkinen
Keyword(s):  
Pinus Sylvestris ◽  
Scots Pine ◽  
Molecular Basis ◽  
Pcr Primers ◽  
Pollen Grain ◽  
Plant Genome ◽  
Oligonucleotide Probes ◽  
Genomic Libraries ◽  
Pcr Products

Scots pine (Pinus sylvestris) genomic libraries were constructed and screened with oligonucleotide probes (GT)10, (CT)10, and (AT)10. Eight microsatellites were identified from 6000 clones screened. The longest microsatellite stretch found, (GT)9(N)21(AT)24, was amplified from bud and single pollen grain samples. In order to clarify the complex amplification pattern revealed, two PCR products were sequenced. The size differences were caused both by varying repeat numbers of the microsatellite stretches and by differences in other parts of the amplified sequence. This kind of complex molecular basis of microsatellite amplification within a species has not been previously reported. Microsatellite sequences were used as PCR primers to detect polymorphisms and to estimate the abundance of microsatellites.Key words: microsatellites, Pinus sylvestris, plant genome, inter SSR–PCR.

Regulatory elements controlling pituitary-specific expression of the human prolactin gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4690-4700
Author(s):  
B Peers ◽  
M L Voz ◽  
P Monget ◽  
M Mathy-Hartert ◽  
M Berwaer ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Distal Region ◽  
Dnase I ◽  
Proximal Region ◽  
Base Pairs ◽  
Specific Expression ◽  
Positive Region ◽  
Dnase I Footprinting ◽  
Human Prolactin ◽  
Flanking Sequences

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice

1988 ◽  
Vol 8 (4) ◽  
pp. 1821-1825
Author(s):  
K A Kelley ◽  
J W Chamberlain ◽  
J A Nolan ◽  
A L Horwich ◽  
F Kalousek ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Transgenic Animals ◽  
Ornithine Transcarbamylase ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Protein Coding ◽  
Wide Range ◽  
Flanking Sequences

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.

scholarly journals Detection of erythromycin-resistant determinants by PCR.

1996 ◽  
Vol 40 (11) ◽  
pp. 2562-2566 ◽  
Author(s):  
J Sutcliffe ◽  
T Grebe ◽  
A Tait-Kamradt ◽  
L Wondrack
Keyword(s):  
Direct Sequencing ◽  
Pcr Primers ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Gene Products ◽  
Mechanisms Of Resistance ◽  
Erythromycin Resistance ◽  
Surveillance Studies ◽  
Resistance Determinants ◽  
Pcr Products

Erythromycin resistance determinants include Erm methylases, efflux pumps, and inactivating enzymes. To distinguish the different mechanisms of resistance in clinical isolates, PCR primers were designed so that amplification of the partial gene products could be detected in multiplex PCRs. This methodology enables the direct sequencing of amplified PCR products that can be used to compare resistance determinants in clinical strains. Further, this methodology could be useful in surveillance studies of erythromycin-resistant determinants.

scholarly journals PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

1999 ◽  
Vol 65 (4) ◽  
pp. 1652-1657 ◽  
Author(s):  
Sara Hallin ◽  
Per-Eric Lindgren
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Nitrite Reductase ◽  
Wastewater Treatment Plants ◽  
Paracoccus Denitrificans ◽  
Pcr Primers ◽  
Genes Encoding ◽  
Pcr Products ◽  
Key Enzyme ◽  
Sequence Relationships

ABSTRACT Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd 1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnir genes were also established. Thecd 1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

scholarly journals Detection and Differentiation of Primate α-herpesviruses by PCR

1997 ◽  
Vol 9 (3) ◽  
pp. 225-231 ◽  
Author(s):  
Daria H. Black ◽  
R. Eberle
Keyword(s):  
Pcr Primers ◽  
Restriction Pattern ◽  
Restriction Enzyme Digestion ◽  
Gene Encoding ◽  
B Virus ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Unique Restriction ◽  
Polymerase Chain ◽  
Simplex Virus

A rapid method for detection and differentiation of 5 primate αpha-herpesviruses (human herpes simplex virus types 1 and 2 [HSV1, HSV2], green monkey simian agent 8, baboon herpesvirus 2 [HVP2], and macaque B virus [BV]) was developed utilizing the polymerase chain reaction (PCR). PCR primers were located in conserved regions of the gene encoding the glycoprotein B, which flanks an intervening region that is highly divergent among the 5 viruses. Amplified PCR products from the 5 viruses were readily differentiated by their unique restriction enzyme digestion patterns. No variation in digestion patterns was noted among strains of HSV1, HSV2, or HVP2. One clinical isolate of BV exhibited variation in a single restriction site, but its overall restriction pattern remained typical of BV. This method (PCR/RFLP) allowed the presence of herpesvirus DNA in clinical swabs from primates to be readily detected and the virus unambiguously identified.

scholarly journals Functional analysis of elements affecting expression of the beta-actin gene of carp.

1990 ◽  
Vol 10 (7) ◽  
pp. 3432-3440 ◽  
Author(s):  
Z J Liu ◽  
B Moav ◽  
A J Faras ◽  
K S Guise ◽  
A R Kapuscinski ◽  
...  
Keyword(s):  
Regulatory Region ◽  
Housekeeping Genes ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Actin Gene ◽  
Base Pairs ◽  
First Intron ◽  
Beta Actin ◽  
Flanking Sequences ◽  
Mouse Cells

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.

Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products

2014 ◽  
Vol 565 ◽  
pp. 3-8 ◽  
Author(s):  
Ji Gang Li ◽  
Guo Ying Han ◽  
Xiu Min Li ◽  
Jiao Jiao Sun ◽  
Ke Jing Song ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Pcr Primers ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reaction Products ◽  
Major Disadvantage ◽  
Cloning Method ◽  
Directional Cloning ◽  
Pcr Products ◽  
Green Fluorescent

Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.

scholarly journals Specific PCR detection of Peptostreptococcus magnus

2003 ◽  
Vol 52 (4) ◽  
pp. 309-313 ◽  
Author(s):  
M.P. Riggio ◽  
A. Lennon
Keyword(s):  
Pcr Primers ◽  
Oral Bacteria ◽  
Rrna Gene ◽  
Subgingival Plaque ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Specific Pcr ◽  
Wide Range ◽  
Pcr Products ◽  
Peptostreptococcus Magnus

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3′ ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

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