Quantitative isolation of DNA restriction fragments from low-melting agarose by Elutip-d affinity chromatography

Schmitt, John J.; Cohen, Bennett N.

doi:10.1016/0003-2697(83)90109-4

Cited by 56 publications

(13 citation statements)

References 5 publications

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“…The ClaI-cut vector was treated with calf intestinal alkaline phosphatase to prevent self-ligation (13). The appropriate Clal fragment of pMK3 was isolated from a 1% low-meltingpoint agarose gel and purified on an Elutip-D column (22). The fragment was ligated to the cut vector, and the ligation mix was used to transform RR1 cells to ampicillin resistance (13).…”

Section: Methodsmentioning

confidence: 99%

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Kurtz¹,

Cortelyou²,

Kirsch³

1986

Mol. Cell. Biol.

163

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Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.

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Section: Methodsmentioning

confidence: 99%

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Kurtz¹,

Cortelyou²,

Kirsch³

1986

Mol. Cell. Biol.

163

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“…The fragments were separated on 1% low melting temperature agarose (Seaplaque). The DNA bands were recovered from the gel slices (22) and ligated into the vectors described in Fig. 1.…”

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confidence: 99%

Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.

Williams¹,

Fox²,

Shea³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated H1MaI, comprises lipid and two membrane proteins, ll-AM and II-BMaI. The proteins are encoded by ptsM, located at "40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for X DNA penetration of the cytoplasmic membrane. Earlier studies suggested that bothpel function and ll-Bm are encoded by the same gene, while a different gene (also in ptsM) encodes II-AM". In the present studies, a ptsM done, pCS13, was isolated from an E. colU Hindml gene bank in pBR322 and restored both mannose fermentation and pelt function to ptsM mutants defective in II-BMaI. Subclones of pCS13 show that (i) two distinct genes, manY and manZ, encode thepel function and the II-BMa`protein, respectively; (ii) each gene may have its own promoter; (iii) whereas the protein encoded by manY (Pel) alone seems sufficient for X sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the HIMan complex. The efficiency of the complex may depend on the ratio of Pel to 11Man.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the translocation of its sugar substrates across the bacterial membrane concomitant with their phosphorylation (1-3). The PTS consists of two cytoplasmic phosphorylated carrier proteins, enzyme I and HPr (which are not sugar specific) and a number of sugar-specific membrane-associated proteins, called enzyme II complexes. Phosphoryl group transfer occurs in the following sequence: phosphoenolpyruvate -* enzyme I --HPr --enzyme II sugar.Two enzyme II complexes catalyze glucose translocation across the Escherichia coli membrane (4, 5). One, designated IIGlc, is specific for glucose and methyl glucosides. The second complex, IIMan, the subject of this report, comprises two membrane-associated proteins II-AMan and II-BMan, and phosphorylates glucose, mannose, and their analogues, such as 2-deoxyglucose (4-7).The concept of two proteins in the IIMan complex was based on early biochemical data (7). By contrast, genetic mapping suggested only a single locus for ptsM (8), at =40 minutes on the E. coli map. Another mutation in this region, called pel, resulted in resistance to penetration of the bacterial inner membrane by X DNA (9, 10). pel appeared to be closely related to ptsM since strains bearing mutations in pel were unable to ferment mannose (although only 30% of the strains unable to ferment mannose were insensitive to X phage).Recent work in this and other laboratories (refs. 11 and 12; P. Saris and E. T. Palva, personal communication) has shown thatptsM contains two structural genes-one encodes II-AMan (manX), and the other encodes II-BMan (manZ). Transcription occurs from manX through manZ with a str...

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“…At various times after induction of flagellation, RNA was isolated from cells by the modified guanidinium thiocyanate method outlined by Burland et al (2) (19), except that after fragment isolation the suspended DNA was extracted twice each with phenol and chloroform-isoamyl alcohol, precipitated, and resuspended. DNA was nick translated by the method of Rigby et al (14).…”

mentioning

confidence: 99%

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Green¹,

Dove²

1984

Mol. Cell. Biol.

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Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, ca3, that is not found in nonflagellated cells. Evidence is presented that suggests that a3 tubulin arises through posttranslational modification of a preexisting a tubulin. Pulse-chase experiments showed that labeled a3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no 03 tubulin, also consistent with a3 being made by posttranslational modification. Levels of a and ,I tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.Physarum polycephalum expresses multiple forms of tubulin through its life cycle. Two-dimensional (2D) gel electrophoresis of myxamoebal proteins resolves otl and 31 tubulin spots. When myxamoebae are induced to become flagellates, a new spot appears in the tubulin region of 2D gels of cellular proteins. Peptide mapping confirms that this spot is composed of an a tubulin. The appearance of this new tubulin, a3, is correlated with the presence of flagella (2).In vitro translations of total RNA from axenically grown myxamoebae, which do not flagellate, reveal no a3 tubulin in the protein products, as is the case for in vitro translations of RNA from Physarum plasmodia (2). This information suggests that there is not a constitutively expressed RNA for a3. These data, coupled with the relatively rapid appearance of a3 tubulin after induction of flagellation, have led Burland et al. (2) to suggest that a3 tubulin is made via a posttranslational modification of al tubulin. The flagellum-specific a tubulin of Chlamydomonas reinhardtii is made by posttranslational modification of an a tubulin (9), and it has also been reported that the flagellar a tubulin of the trypanosome Crithidia fasciculata is posttranslationally modified (17).Changes in levels of tubulin RNAs occur after deflagellation of Chlamydomonas reinhardtii (13, 18), deciliation of Tetrahymena pyriformis (6,10) potassium orthophosphate, 0.025% sodium chloride, 0.021% magnesium sulfate, 0.005% calcium chloride in distilled water, pH 5.0 with potassium hydroxide). A suspension of myxamoebae in basal salts solution was diluted to a starting density of ca. 5 x 105 myxamoebae per ml, and one-tenth of this volume of the concentrated E. coli was added. This suspension was transferred to glass or plastic petri plates to give about 1 ml of suspension per 6-cm2 surface area. All incubations were stationary at 26°C.In vivo labeling of polypeptides. Myxamoebal proteins were labeled by adding 50 ,uCi of [35S]methionine (SJ204;Amersham Corp., Arlington Heights, Ill.) per ml of liquid culture at the time of preparation of the culture. Myxamoebae grown by this method settle on the bottom of the plate so they must be resuspe...

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Quantitative isolation of DNA restriction fragments from low-melting agarose by Elutip-d affinity chromatography

Cited by 56 publications

References 5 publications

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

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