Selection Of Specific Clones Research Articles

Genotypic variability and stability of poplars and willows grown onnitrate-contaminated soils

Phyto-recurrent selection is an established method for selecting tree genotypes for phytoremediation. To identify promising Populus (poplar) and Salix (willow) genotypes for phytotechnologies, our objectives were to (1) evaluate the genotypic variability in survival, height, and diameter of poplar and willow clones established on soils heavily contaminated with nitrates; and (2) assess the genotypic stability in survival and diameter of selected poplar clones after one and eleven growing seasons. We tested 27 poplar and 10 willow clones planted as unrooted cuttings, along with 15 poplar genotypes planted as rooted cuttings. The trees were tested at an agricultural production facility in the Midwestern, United States. After 11 growing seasons, using phyto-recurrent selection, we surveyed survival and measured the diameter of 27 poplar clones (14 unrooted, 13 rooted) that were selected based on superior survival and growth throughout plantation development. Overall, willow exhibited the greatest survival, while poplar had the greatest height and diameter. At 11 years after planting, superior clones were identified that exhibited above-average diameter growth at the establishment- and rotation-age, most of which had stable genotypic performance over time. Selection of specific clones was favorable to genomic groups, based on the geographic location and soil conditions of the site.

Epidemiology of Shigellosis in Korea

Shigellosis is an acute diarrheal disease caused by bacteria of the genus Shigella. Following the occurrence of a large outbreak of shigellosis as well as sporadic cases since 1998, shigellosis has been a major health problem in Korea. There have been major changes in epidemiology during the last five decades concerning shigellosis in terms of total incidence of shigellosis, prevalence of certain serogroups, selection of specific clones, and introduction of new Shigella clones. S. dysenteriae was the most prevalent species until the early twentieth century, S. flexneri was the most prevalent until the late 1980s, and S. sonnei has been the most prevalent since 1990. Diverse serotypes of S. dysenteriae (4 serotypes), S. flexneri (8 serotypes), and S. boydii (4 serotypes) were found during the Korean War and many of these Korean endemic Shigella strains circulated in the community until the late 1970s. However, the endemic strains of S. dysenteriae, S. boydii, and S. sonnei disappeared in the late 1980s. A new clone of S. sonnei that was introduced between the late 1980s and the early 1990s was responsible for a large proportion of shigellosis in recent years. S. flexneri serotype 4a was the most frequently found during the Korean War and then the incidence of S. flexneri 2a gradually increased with time. S. flexneri isolates detected from 1991 to 1997 were all serotype 2a. However, the diverse clones of S. flexneri reemerged in Korea since 1999. It has not been determined whether the S. flexneri strains from the 2000s were the descendants of the Korean endemic strains or imported new strains, but the PFGE patterns were different between S. flexneri strains from the 1980s and 2000s. The widespread of new S. sonnei strains and the persistence of S. flexneri strains are responsible for the endemicity of shigellosis in Korea.

FLT3/Internal Tandem Duplication in Paired Presentation and Relapse Pediatric AML Samples.

We have previously reported that FLT3 internal tandem duplications (FLT3/ITD) were detectable in 11.5% of 234 pediatric AML samples at initial diagnoses, and conferred a poor prognosis (Zwaan et al, Blood 2003). However, no data were available on relapsed pediatric AML and on paired initial-relapse samples. Currently we have studied genomic DNA of 99 relapsed and 42 paired initial-relapse pediatric AML samples for FLT3/ITD. Samples were obtained from the AML-BFM-SG, the Dutch Childhood Oncology Group, and by centers participating in the International BFM Relapsed AML 2001/01 study.Of the 99 relapsed patients the median age was 10.2 years and 71% were boys. Median time from diagnosis to first relapse was 14 months. Patient characteristics of the 42 pairs were at diagnosis: median age 7.7 years, sex 80% boys; at relapse: median age 14.3 years. Median time from diagnosis to first relapse was 14 months. ITDs were detected by PCR amplification including a fluorescent labeled primer. Fragments were separated by capillary electrophoresis on an ABI 3100 and analyzed using Genescan software. Of the 99 relapsed samples, 20 were ITD positive (20.2%), with ITDs varying in length from 18–129 bp. ITDs occurred in 15% of children <10 years of age, and in 21% ≥ 10 years (p=0.5). ITD pos. patients were boys in 84% vs. 68% in wild type (wt) patients (p=0.16). WBC, blast percentage and FAB-types were not different between ITD and wt patients. The time-interval between diagnosis and relapse was significantly shorter in ITD pos. patients (10 months) than in wt ones (15 months, p=0.003).Flt3/ITD in paired initial vs relapse samples of pediatric AMLRelapse ITD posRelapse wild typeTotalInitial ITD pos6 (14%)4 (10%)10 (24%)Initial wild type4 (10%)28 (66%)32 (76%)Total10 (24%)32 (76%)42 (100%)Two of the 42 paired samples had 2 different length mutations at initial diagnosis and only 1 at relapse. Four acquired an ITD at relapse (one at 2nd relapse), which varied from 21–129 bp. FAB-types were: 1xM1, 2xM2, 1x M6. Median time to first relapse was only 6 months. Four patients (FAB-types were 2xM1 and 2xM2) lost the ITD, which at diagnosis varied from 30–72 bp and interestingly the median time to relapse was relatively long (median of 30 months). All ITDs were confirmed by sequencing and they were all located in exon 14. We conclude that: a) detectable FLT3/ITD is more frequent in the relapsed patients (±20%) compared to our previous study with patients at initial diagnosis (11.5%); b) patients with initially detectable FLT3/ITD relapse earlier than wt patients; c) there were no significant differences in clinical and cell-biological characteristics between wt and ITD patients at relapse d) gain and loss of ITDs at relapse does occur e) patients who gain an ITD seem to relapse early, patients who lose an ITD relapse relatively late. The changes in ITD status may reflect true de novo mutations or a selection of specific clones. In the latter case a small ITD positive subclone was not detected. We will further elucidate this with specific PCRs we are currently developing. These results have relevance for Flt3-targeted therapy and minimal disease monitoring.

Finding new genes in the nervous system by cDNA analysis

There are several methods to isolate c DNAs that do not require prior biochemical characterization of their protein products, including isolation of new members of particular protein families based on sequences of known members, selection of specific clones by differential screening, and shotgun analysis of a particular c DNA population. This third approach will produce a huge catalogue of c DNA sequences and anatomical data that will be useful to investigators with diverse interests. Recent methodological advances allow c DNAs to be isolated and sequenced rapidly. These methods, coupled with fast and reliable techniques for in situ hybridization histochemistry, should permit c DNA catalogues to be assembled efficiently.

Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion.

Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice. Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8-azaguanine-resistant clone (X63-Ag8) of MOPC 21 myeloma. Over 50% of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10% of the hybrid lines exhibit anti-SRBC activity. The high proportion of antibody-producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production. In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1-Ag4-1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains. Three anti-SRBC (probably of the mu, gamma2b and gamma1 classes, respectively) and two anti-2,4,6-trinitrophenyl (of the mu class) antibody-producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma gamma and K chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.