Direct clone characterization from plaques and colonies by the polymerase chain reaction

Güssow, Detlef; Clackson, Tim

doi:10.1093/nar/17.10.4000

Cited by 299 publications

(126 citation statements)

References 1 publication

Supporting

Mentioning

123

Contrasting

Unclassified

Order By: Relevance

“…Direct PCR (13) performed in the presence of the primers pM20-deg (100 pmol) and pT7 (20 pmol, 5Ј-AATACGACTCACTATAGGGC-3Ј), the latter corresponding to the T7 promoter of the -Zap II DNA and 2 ϫ 10 5 plaque-forming units from an M. sexta larval midgut -Zap II cDNA library (14). The reaction was carried out with AmpliTaq DNA polymerase (PerkinElmer) in a buffer consisting of 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin, 200 M of each dNTP, and 10 mM Tris-HCl (pH 8.3).…”

Section: Methodsmentioning

confidence: 99%

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Merzendorfer

Huss

Schmid

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal antibody to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptidespecific monoclonal antibody. Thus, extensive N-glycosylation of the novel V o subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the V o complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta. Hϩ V-ATPases are a class of ion transport proteins that couple ATP hydrolysis to the movement of protons across membranes. In endomembranes they function, in concert with chloride channels, as acidifiers of intracellular compartments, whereas in plasma membranes their roles are dependent on the cell type. V-ATPases consist of a peripheral V 1 complex, which is responsible for the hydrolysis of ATP, and a membranebound V o complex which is responsible for the translocation of protons. Although the subunit composition may depend upon the source of the enzyme, at least seven subunits of the V 1 complex, subunits A to G, appear to be universal V-ATPase components (1). By contrast, the subunit composition of the V o complex is less clear. There is no doubt that a 16 -17-kDa proteolipid, the proton "channel," is a major constituent of the V o complex. A membrane-associated subunit in the 40-kDa range and an ϳ100-kDa transmembrane subunit may be two additional essential V o components (1). Recently, a novel 9.2-kDa membrane sector-associated polypeptide was reported from bovine chromaffin granules (2). Its sequence and structure show some similarity to Vma21p, a yeast protein involved in the assembly of the V-ATPase; whether or not it is a constitutive V-ATPase subunit remains an open question.In the larval midgut epithelium of the model insect, Manduca sexta (Lepidoptera, Sphingidae), a plasma membrane V-ATPase is present in the apical membranes of goblet cells where it energizes the alkalinization of the gut lumen to a pH of more than 11 (3). For the V 1 complex, amino acid sequences of five insect subunits A, B, E, F, and G have been deduced from cloned cDNAs (4), and evidence for the existence of subuni...

show abstract

Section: Methodsmentioning

confidence: 99%

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Merzendorfer

Huss

Schmid

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The genomic copy number of 272 clones from the library was determined. The cloned insert size was estimated by PCR amplification of the recombinant DNA using M13 sequencing primers (Gussow & Clackson 1989). The average insert size was approx-imately 1000 base pairs (bp) in length.…”

Section: Materials and Methods (A) Marker Isolationmentioning

confidence: 99%

Reconstructing labroid evolution with single–copy nuclear DNA

Streelman

Karl

1997

Proc. R. Soc. Lond. B

113

View full text Add to dashboard Cite

SUMMARYFifteen per cent of all living fishes are united in a single suborder (Labroidei) and display a dazzling array of behavioural and ecological traits. The labroids are considered monophyletic and members share a pharyngeal jaw apparatus (PJA) modified for crushing and processing prey. Outside of the explicitly functional PJA, there is no corroborative evidence for a monophyletic Labroidei. Here, we report the first molecular phylogenetic analysis of the suborder. Contrary to morphology-based phylogenies, our single-copy nuclear DNA data do not support labroid families as a natural group. Our data indicate that pharyngognathy has evolved independently among labroid families and that characters of the PJA are not reliable markers of perciform evolution. This work 'crushes' conventional views of fish phylogeny and should engender novel concepts of piscine life history evolution.

show abstract

“…The clones were initially screened by colony PCR (Gussow and Clackson, 1989), followed by digestion of cloned sequences using the restriction enzyme MvnI (Roche Diagnostics Corp., Indianapolis, IN, USA). Digests were loaded on 2% agarose gels to evaluate restriction patterns.…”

Section: Study Site and Sample Collectionmentioning

confidence: 99%

Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes

et al. 2007

View full text Add to dashboard Cite

Anopheles gambiae mosquitoes are not known to harbor endosymbiotic bacteria. Here we show, using nucleic acid-based methods, that 16S rRNA gene sequences specific to a recently described mosquito midgut bacterium, Thorsellia anophelis, is predominant in the midgut of adult An. gambiae s.l. mosquitoes captured in residences in central Kenya, and also occurs in the aquatic rice paddy environment nearby. PCR consistently detected T. anophelis in the surface microlayer of rice paddies, which is also consistent with the surface-feeding behavior of A. gambiae s.l. larvae. Phylogenetic analysis of cloned environmental 16S rRNA genes identified four major Thorsellia lineages, which are closely affiliated to an insect endosymbiont of the genus Arsenophonus. Physiological characterizations support the hypothesis that T. anophelis is well adapted to the female anopheline midgut by utilizing blood and tolerating the alkaline conditions in this environment. The results suggest that aquatically derived bacteria such as T. anophelis can persist through mosquito metamorphosis and become well-established in the adult mosquito midgut.

show abstract

Direct clone characterization from plaques and colonies by the polymerase chain reaction

Cited by 299 publications

References 1 publication

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Reconstructing labroid evolution with single–copy nuclear DNA

Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes

Contact Info

Product

Resources

About