Gene Fingerprinting Research Articles

Direct Classification and Selection of Superior Alleles for Crop Improvement

The use of conventional breeding methods has resulted in consistent crop improvement within the cultivated gene pool by producing genotypes with new combinations of alleles that produce better phenotypes than either of the parents (transgressive segregation). Biotechnology has provided new methods to generate, identify, characterize, and manipulate genetic variation. Among these methods, marker assisted selection (MAS) with DNA markers has been applied in plant improvement programs. However, MAS is limited by the effort required to generate information about map location and breeding value of genes controlling important traits. Comparative genetic analysis across the domesticated grasses facilitates the identification and localization of gene sequences controlling specific traits of interest. The emerging databases of gene sequences will allow directed discovery of genes in higher plants and classification of alleles present within breeding germplasm. Identification of the genes controlling a trait and knowledge of their DNA sequence would facilitate classification of variation in the germplasm pool based on gene fingerprinting or characterization of variation in key DNA sequences. Classification of sequence variants at a targeted locus would substantially reduce the amount of work required to determine their relative breeding value and lead to the identification of superior alleles. Combining direct allele selection (DAS) with conventional selection, would allow more rapid and precise improvement of populations and breeding lines. Limitations of current technology can be minimized by transfer of genetic information across species, identification of highly variable genes, and focusing on the most important genes and traits for the species of interest.

Intraclonal mating in Trypanosoma brucei is associated with out-crossing.

To examine whether mating can occur within as well as between clones of Trypanosoma brucei, we transformed three T. brucei subspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter- and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intra- as well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase THT1 glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.

Three Predominant Clones Identified Within Penicillin-Resistant South African Isolates of Streptococcus pneumoniae

The worldwide spread of antibiotic-resistant pneumococcal clones is cause for concern. In South Africa, penicillin-resistant pneumococci are mostly associated with serogroups 6 and 19, therefore, we have analyzed the clonal relationships between resistant isolates of these two serogroups. DNA fingerprinting of penicillin-binding protein genes and arbitrarily-primed PCR, have identified two penicillin-resistant pneumococcal clones of serotype 19A and serotype 6B, and a third multi-resistant serotype 19A clone, of which all three clones are predominant within South Africa's penicillin-resistant pneumococcal population. These clones are represented by strains isolated from hospitals that are widely separated in South Africa. Further studies are required to establish whether these clones have expanded in the rest of Africa or spread to other continents.

Molecular detection of clonally rearranged cells in peripheral blood progenitor cell harvests from multiple myeloma patients.

Peripheral blood progenitor cells (PBPC) are increasingly used for autologous reconstitution following high-dose chemotherapy in multiple myeloma but it is unclear whether these cells are less likely to be contaminated with malignant cells than bone marrow (BM). We have investigated this using immunoglobulin heavy-chain (IgH) gene fingerprinting, a polymerase chain reaction based technique with a sensitivity of 0.1-0.01% (10(-3)-10(-4)). We have looked for patient-specific IgH rearrangements in leukapheresis samples from eight myeloma patients undergoing PBPC harvest. Seven were in first remission (six partial, one complete) and one in second complete remission. Mobilization of PBPC was accomplished using cyclophosphamide (4 or 7 mg/m2) and rhG- or GM-CSF. Between two and five leukaphereses were performed in each patient. Patient-specific IgH rearrangements were identified in diagnostic BM in all patients and bands of identical size were found in one or more leukaphereses from 6/8 patients. Overall, 14/32 leukaphereses were shown to be contaminated. Two patients who showed contamination of at least one PBPC harvest had BM harvests in which contaminating cells were not detectable, suggesting that PBPC are not necessarily less likely to be contaminated than marrow stem cells. These results indicate that PBPC harvests from the majority of myeloma patients are likely to contain contaminating cells. Further studies are needed to determine whether these cells are clonogenic and whether they contribute to relapse.

Molecular characterization of the coagulase-negative staphylococcal surface flora of premature neonates

A single point study was conducted to determine which surface sites best represent the density and composition of the coagulase-negative staphylococcal (CNS) colonizing flora in premature neonates. Five different surface sites of six randomly selected neonates hospitalized in a neonatal intensive care unit (NICU) for a month were examined. The individual strains and their clonal organization within CNS species were identified using restriction endonuclease fingerprinting of whole chromosomal DNA and ribosomal RNA genes. Cultures of the scalp, umbilicus, foot, nose and rectum were collected and quantitatively processed. Ten colonies were typed per surface culture. The most dense CNS colonization was noted on the umbilicus (mean 1.2 x 10(4) c.f.u. cm-2), foot (mean 1.6 x 10(3) c.f.u. cm-2) and nose (mean 1.7 x 10(3) c.f.u. cm-2) of NICU neonates. Scalp and rectum were scarcely colonized. Of all the CNS surface isolates, S. epidermidis accounted for 77.7% (219/282) and S. haemolyticus, S. warneri and S. capitis accounted for 20.6% (58/282), 1.4% (4/282) and 0.4% (1/282), respectively. Colonization of each surface site comprised a maximum of five different strains representing four CNS species. Overall, five clones of S. epidermidis, two of S. haemolyticus, one of S. warneri and one of S. capitis were noted among the 282 isolates. The most predominant were two clones of S. epidermidis and one of S. haemolyticus; they accounted for 94% (265/282). Cultures from the foot and scalp represented the most heterogeneous CNS colonization of the five sites examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya.

A total of 26% of the pneumococci isolated from an outpatient clinic in Nairobi, Kenya, during 1991 to 1992 had intermediate levels of penicillin resistance. Gene fingerprinting and DNA sequencing were used to distinguish the penicillin-binding protein (PBP) 1A, 2B, and 2X genes in 23 resistant isolates. Isolates were grouped into those that had identical forms of each of the three PBP genes (fingerprint groups) and those that had identical rRNA gene restriction patterns (ribotypes). Both methods divided the isolates into 11 groups. In a few cases, horizontal gene transfer appeared to have distributed an identical altered PBP gene into different pneumococcal lineages. Eight isolates were indistinguishable by ribotyping or multilocus enzyme electrophoresis and contained identical PBP 1A genes. Although these isolates were therefore members of the same clone, they were divided into two fingerprint groups which contained different PBP 2X and 2B genes. Presumably, members of this clone have acquired different altered PBP 2X and 2B genes on two separate occasions. One of these fingerprint groups contained isolates of serotype 14, whereas the other contained isolates of both serotypes 14 and 7. The identification of isolates in the latter group that are identical by all criteria, except serotype, implies the occurrence of a change in serotype. The predominant serotypes of the penicillin-resistant pneumococci from Nairobi were serotypes 14 and 19. In both cases, isolates of the same serotype which required the same MIC of penicillin were not members of a single clone, indicating that identity of serotype and MIC are not sufficient criteria for defining clones of resistant pneumococci even when the bacteria are isolated from a single clinic.

Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae.

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.

Genetic diversity of penicillin-binding protein 2 genes of penicillin-resistant strains of Neisseria meningitidis revealed by fingerprinting of amplified DNA

A 2-kilobase fragment containing the penicillin-binding protein 2 gene (penA) was amplified by using the polymerase chain reaction with DNA prepared from 35 penicillin-resistant strains of Neisseria meningitidis isolated in England, Ireland, and Spain (MICs, 0.16 to 1.28 micrograms of benzylpenicillin per ml) and from 10 penicillin-susceptible strains (MICs, less than or equal to 0.04 micrograms of benzylpenicillin per ml). The penA genes were digested with HpaII or TaqYI; and the resulting fragments were end-labeled, fractionated on a polyacrylamide gel, and autoradiographed, to produce gene fingerprints. The fingerprints of the penA genes of the 10 penicillin-susceptible strains were identical. In contrast, the fingerprints of the penA genes of all but two of the penicillin-resistant strains differed markedly from those of the penicillin-susceptible strains. The altered penA genes of group B penicillin-resistant strains were very diverse, and 14 different fingerprints were found among the 15 strains that we examined. The penA genes of the 19 resistant group C strains were less diverse, and two major fingerprint patterns predominated.