Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

FINGAR: A new genetic algorithm-based method for fitting NMR data

Summary A new NMR refinement method, FINGAR (FIt NMR using a Genetic AlgoRithm), has been developed, which allows one to determine a weighted set of structures that best fits measured NMR-derived data. This method shows appreciable advantages over commonly used refinement methods. FINGAR generates an ensemble of conformations whose average reproduces the experimental NMR-derived restraints. In addition, a statistical importance weight is assigned to each of the conformations in the ensemble. As a result, one is not limited to simply presenting an envelope of sampled conformers. Instead, one can subsequently focus on a select few conformers of high weight. This is critical, because many structural analyses depend on using discrete conformations, not simply averages or ensembles. The genetic algorithm used by FINGAR allows one to simultaneously and reliably fit against many restraints, and to generate solutions which include as many conformations with non-zero weights as are necessary to generate the best fit. An added benefit of FINGAR is that because the time-consuming step in this method needs only to be performed once, in the beginning of the first run, numerous FINGAR simulations can be performed rapidly.     

Correlation of endoscopic cytology and histology in oesophageal cancer: results in Porto Alegre,RS—Brazil

A retrospective study of oesophageal cytopathology at the Hospital de Clínicas de Porto Alegre (HCPA), RS, Brazil, from 1989 to 1992 was made to assess the sensitivity, specificity, predictive values and accuracy of endoscopic cytology and biopsy; and study the correlation between cytopathological and histopathological diagnosis. Specimens from 94 patients were available for review. The final diagnosis was based on surgical pathology and follow up. The 81 patients with cancer of the oesophagus had the following sex distribution: 64 males and 17 females (a 3.7–1 ratio). No tumour was found in 13 patients. The following conclusions were made: (i) there is excellent correlation between cytology and histology in oesophageal lesions sampled by endoscopy; (ii) a correct positive cytologic report was obtained in 77 (95%) of the 81 proven oesophageal cancers; a false-negative or unsatisfactory result was given in four patients. A false-positive diagnosis of cancer was not made. There were 13 true-negative reports. These findings result in a sensitivity of 95% with 95% confidence intervals (CI) of 90.26–99.74%; a specificity of 100% (CI of 98.5–100%); a positive predictive value of 100% (CI of 99.3–100%); a negative predictive value of 76% (CI of 55.7–96.3%); (iii) a correct positive histological report was obtained in 67 (83%) of the 81 proven oesophageal cancers; a false-negative or unsatisfactory result was given in 14 patients. A false-positive diagnosis of cancer was not made. There were 13 true-negative reports. These findings result in a sensitivity of 83% with 95% CI of 74.82–91.18%; a specificity of 100% (CI of 98.5–100%); a positive predictive value of 100% (CI of 99.25–100%); a negative predictive value of 48% (CI of 29.16–64.84%); (iv) of 81 patients with proven cancer, in 79 (98%) at least one of the methods was positive. In only two patients with cancer were both methods negative. These findings result in a combined sensitivity of 98% (CI of 94.92–100%); a specificity of 100% (CI of 98.5–100%); a positive predictive value of 100% (CI of 99.31–100%); and a negative predictive value of 87% (CI of 70–100%). Our series confirms the value of the combined use of cytology and biopsy for the investigation of oesophageal lesions. However, it should be remembered that even with the combined use of cytology and biopsy there are some tumours that will be negative by both procedures: we had only two such cases, confirming the rarity of such an event.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Quantitative trait analysis of fruit quality in cucumber: QTL detection,confirmation, and comparison with mating-design variation

A cross within C. sativus var. sativus (GY14 x P1432860) and molecular markers were used to determine the number, magnitudes of effect, and overall variation described for genes conditioning the quantitatively inherited traits of length, diameter, seed-cavity size, color, L/D (length/diameter), and S/D (seed-cavity size/diameter). QTL effects were detected with MAPMAKER/QTL using 100 F₃ lines evaluated in a replicated field trial of two harvests over 2 years at one location. Multilocus models were constructed by fixing significant intervals and re-scanning using MAPMAKER/ QTL. Marker inclusion in multilocus models was compared to an ANOVA backward elimination procedure. Generally the same loci were associated with QTLs among the two methods of model construction. Heritabilities of individual QTLs were confirmed by analysis of related backcrosses (67 BC₁P₁ lines and 68 BC₁ P₂ lines). The majority of QTLs were confirmed in at least one backcross population. Pairs of backcrosses allowed overall additive variances and heritabilities to be calculated using a North Carolina Design III (NCIII design) and estimates were compared to overall variances attributable to markers. Heritability estimates using markers were comparable, but generally lower than additive variances estimated by co-variance relationships in the NCIII design. This suggests that neither the number nor the magnitude of QTL effects were overestimated. The utility of backcrosses to confirm individual QTLs and the overall variance described by QTLs is recommended to avoid false positives and over-estimation of effects. The number of QTLs, and/or the proportions of phenotypic variation described by markers and the mating design, agreed with previous reports of heritabilities employing similar germplasm.     

Structural evolution of wheat chromosomes 4A, 5A,and 7B and its impact on recombination

The construction of comparative genetic maps of chromosomes 4A^m and 5A^m of Triticum monococcum and chromosomes of homoeologous groups 4, 5 and 7 of T. aestivum has provided insight into the evolution of these chromosomes. The structures of chromosomes 4A, 5A and 7B of modern-day hexaploid bread wheat can be explained by a 4AL/5AL translocation that occurred at the diploid level and is present both in T. monococcum and T. aestivum. Three further rearrangements, a 4AL/7BS translocation, a pericentric inversion and a paracentric inversion, have taken place in the tetraploid progenitor of hexaploid wheat. These structural rearrangements and the evolution of chromosomes 4A, 5A and 7B of bread wheat are discussed. The presence of the 4AL/5AL translocation in several Triticeae genomes raises two questions — which state is the more primitive, and is the translocation of mono- or poly-phylogenetic origin? The rearrangements that have occurred in chromosome 4A resulted in segments of both arms having different positions relative to the telomere, compared to 4A^m and to 4B and 4D. Comparisons of map length in these regions indicate that genetic length is a function of distance from the telomere, with the distal regions showing the highest recombination.     

A linkage map with RFLP and isozyme markers for almond

Inheritance and linkage studies were conducted with seven isozyme genes and 120 RFLPs in the F₁ progeny of a cross between almond cultivars Ferragnes and Tuono. RFLPs were detected using 57 genomic and 43 cDNA almond clones. Eight of the cDNA probes corresponded to known genes (extensin, prunin (2), -tubulin, endopolygalacturonase, oleosin, actin depolymerizing factor and phosphoglyceromutase). Single-copy clones were found more frequently in the cDNA (65%) than in the genomic libraries (26%). Two maps were elaborated, one with the 93 loci heterozygous in Ferragnes and another with the 69 loci heterozygous in Tuono. Thirty-five loci were heterozygous in both parents and were used as bridges between both maps. Most of the segregations (91%) were of the 11 or 1111 types, and data were analyzed as if they derived from two backcross populations. Eight linkage groups covering 393 cM in Ferragnes and 394 in Tuono were found for each map. None of the loci examined in either map was found to be unlinked. Distorted segregation ratios were mainly concentrated in two linkage groups of the Ferragnes map.     

Maximum-likelihood models for mapping genetic markers showing segregation distortion. 2. F2 populations

In F₂ populations, gametic and zygotic selection may affect the analysis of linkage in different ways. Therefore, specific likelihood equations have to be developed for each case, including dominant and codominant markers. The asymptotic bias of the classical estimates are derived for each case, in order to compare them with the standard errors of the suggested estimates. We discuss the utility and the efficiency of a previous model developed for dominant markers. We show that dominant markers provide very poor information in the case of segregation distortion and, therefore, should be used with circumspection. On the other hand, the estimation of recombination fractions between codominant markers is less affected by selection than is that for dominant markers. We also discuss the analysis of linkage between dominant and codominant markers.