Restriction Fragment Banding Patterns Research Articles

Microbial community development in a laboratory-scale nitrifying activated sludge system with input from a side-stream bioreactor treating digester supernatant

Three laboratory-scale activated sludge treatment trains were operated to investigate the effect on biodiversity in plug flow (PFR) main-stream sewage treatment from input of biomass from side-stream reactors treating anaerobic digester supernatant. One train had a completely mixed (CSTR) side-stream reactor, one a PFR side-stream reactor, and the third train was a control that did not receive input from a side-stream reactor. Restriction endonucleases were used to digest polymerase chain reaction-amplified ammonia monooxygenase subunit A (amoA) genes in monthly samples from each reactor. Restriction fragment banding patterns from polyacrylimide gel electrophoresis indicated that the structure of the ammonia oxidizing bacteria (AOB) populations in all five reactors stabilized by the fourth month of operation and then did not vary subsequently. Furthermore, a dendrogram generated using the Jaccard distance showed that the AOB in each side-stream reactor was most similar to the main-stream reactor in the same train indicating that the AOB population in the side-stream reactor exerts a strong influence on the population in the main-stream reactor. Sequencing results indicated that Nitrosomonas europea, an r-strategist, was the dominant AOB in the PFR side-stream reactor, while Nitrosomonas europea and Nitrosomonas marina, a marine bacterium, were strongly represented in the CSTR side-stream reactor.

Distinguishing the parasitic wasp, Peristenus howardi, from some of its congeners using polymerase chain reaction and restriction endonuclease digestion

A molecular procedure incorporating polymerase chain reaction (PCR) of the COI gene and restriction endonuclease digestion of PCR products was used to distinguish Peristenus howardi (Hymenoptera: Braconidae) from four other Peristenus species. Non-solvent extraction of parasite DNA using a commercially available kit proved to be very effective in producing amplifiable template. Use of SfcI endonuclease produced restriction fragments with banding patterns in agarose gel electrophoresis that readily separated P. howardi, P. digoneutis, P. conradi, P. pallipes, and P. pseudopallipes. However, while the restriction fragment banding patterns of both P. pallipes and P. pseudopallipes were easily distinguishable from the other Peristenus species, they could not be reliably separated from one another. This molecular procedure can be used in applied and ecological research to better understand the role of P. howardi in the Peristenus-Lygus parasite-host system within the Pacific Northwest. Consensus sequences of our amplimers for all five Peristenus spp. are deposited in GenBank under accession numbers AY626370, AY626371, AY626372, AY626373, and AY626374.

Distinguishing the parasitic wasp, Peristenus howardi, from some of its congeners using polymerase chain reaction and restriction endonuclease digestion

A molecular procedure incorporating polymerase chain reaction (PCR) of the COI gene and restriction endonuclease digestion of PCR products was used to distinguish Peristenus howardi (Hymenoptera: Braconidae) from four other Peristenus species. Non-solvent extraction of parasite DNA using a commercially available kit proved to be very effective in producing amplifiable template. Use of SfcI endonuclease produced restriction fragments with banding patterns in agarose gel electrophoresis that readily separated P. howardi, P. digoneutis, P. conradi, P. pallipes, and P. pseudopallipes. However, while the restriction fragment banding patterns of both P. pallipes and P. pseudopallipes were easily distinguishable from the other Peristenus species, they could not be reliably separated from one another. This molecular procedure can be used in applied and ecological research to better understand the role of P. howardi in the Peristenus-Lygus parasite-host system within the Pacific Northwest. Consensus sequences of our amplimers for all five Peristenus spp. are deposited in GenBank.

Molecular Genetic Key for the Identification of 17 Ixodes Species of the United States (Acari: Ixodidae): A Methods Model

A taxonomic key, based on restriction enzyme analysis of the second internal-transcribed spacer (ITS-2) in the nuclear ribosomal DNA gene, was developed for identification of 17 Ixodes tick species in the United States. This key includes: Ixodes affinis Neumann, Ixodes angustus Neumann, Ixodes baergi Cooley and Kohls, Ixodes brunneus Koch, Ixodes cookei Packard, Ixodes dentatus Marx, Ixodes jellisoni Cooley and Kohls, Ixodes kingi Bishopp, Ixodes minor Neumann, Ixodes muris Bishopp and Smith, Ixodes pacificus Cooley and Kohls, Ixodes scapularis Say, Ixodes sculpularis Neumann, I. spinipalpis Hadwen and Nuttall, Ixodes texanus Banks, Ixodes uriae White, and Ixodes woodi Bishopp. A 900-bp fragment of the ITS-2 was amplified using the polymerase chain reaction. This fragment was then digested with the restriction enzymes MspI and CfoI, and the digested fragments were size fractionated on a 2.5% high-resolution agarose gel. A dichotomous key was developed based on digested fragment sizes relative to a standard set of size markers. Little intraspecific variation in restriction fragment banding patterns was detected.

Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate

Mouse lymphoma cells of the L5178Y TK +/−3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Identification of Xanthomonas campestris pv. Juglandis from (Persian) English Walnut Nursery Trees in South Africa

AbstractBacterial isolates producing yellowish colonies on Nutrient Agar were recovered from symptoms of suspect walnut blight disease on leaves of nursery trees in the southwestern Cape Province of South Africa. The isolates were identified by pathogenicity tests on leaves of walnut and plum trees in the greenhouse. Fifteen isolates from four cultivars at two nurseries produced typical lesions of blight on walnut and one isolate. typical lesions of bacterial spot disease on plum leaves. Cluster analysis was done on 28 characteristics recorded from colony growth. colour. form. and elevation on four different culture media, and starch hydrolysis on a semi‐selective medium for the isolation of Xanthomonas campestris pv. juglandis. Total DNA of the isolates was digested with restriction endonuclease Spel and resolved by contour‐clamped homogeneous electric field (CHEF) electrophoresis. Two phenotypic clusters were distinguished among the 15 South African and one reference strain of X.c.pv. juglandis at the 54%Ssm level. The isolate which induced disease symptoms on plum grouped with reference strains of Xanthomonas campestris pv. pruni in a third cluster. Two‐thirds of the isolates were not characterized on the semi‐selective medium for X.c. pv. juglandis. DNA restriction fragment banding patterns were similar for most isolates of X.c.juglandis in the same phenotypic cluster. However, DNA banding patterns were non‐distinct for some isolates with similar phenotypic characters. Phenotypic characteristics and DNA restriction fragment banding patterns of the isolates were not correlated with geographical origin or cultivar specificity.

Mitochondrial DNA Diversity of Roanoke River Striped Bass

Abstract The genetic heterogeneity of mitochondrial DNA (mtDNA) in Roanoke River striped bass Morone saxatilis was examined by restriction endonuclease analysis of mtDNA isolated from 32 specimens captured during the 1989 and 1990 spawning runs. Restriction fragment banding patterns based on restriction site polymorphisms revealed six distinct genotypes, designated I–VI, among the 32 sampled individuals. The predominant genotype, designated genotype I, was found in 59% of the individuals sampled, while genotypes defined by Acc I–Nde I (genotype II), Taq I (genotype III), Taq I–Hinf I (genotype IV), Dde I (genotype V), and Spe I (genotype VI) were found in 25%, 7%, 3%, 3%, and 3% of the individuals. Five distinct mtDNA fragment length variants, designated classes A–E, were identified from the same 32 individuals. The number and size of individual length variants agreed with those identified previously in striped bass populations from geographically distant sites. Analysis of the distribution of length vari...

Genetic characterization ofColletotrichum gloeosporioidesisolates obtained from mango

Abstract Most isolates of Colletotrichum gloeosporioides (>80%) obtained from anthracnose lesions on mango leaves, inflorescences and fruits from different regions in Sri Lanka showed the same ribosomal DNA (rDNA) and very similar mitochondrial DNA (mtDNA) restriction fragment banding patterns when genomic DNA was digested with endonuclease EcoR I. This rDNA restriction fragment banding pattern was the same as that revealed by C. gloeosporioides isolates from mango in other tropical countries. Isolates obtained from inflorescences all showed the same rDNA and mtDNA restriction patterns but a few isolates from fruits and leaves from mango grown in mixed orchards in areas of Sri Lanka with two monsoons and year‐round rainfall had rDNA restriction patterns associated with isolates from other fruit crops. This suggested that some cross‐infection onto mango occurs. However, it is suggested that there is a strain of C. gloeosporioides specific to mango and the clonal nature of this strain implies that it has di...

Ribosomal and mitochondrial DNA polymorphisms in Colletotrichum gloeosporioides isolated from tropical fruits

DNA polymorphisms in 38 isolates of Colletotrichum gloeosporioides infecting avocado, banana, mango and papaya were examined using ribosomal DNA (rDNA) from Saccharomyces carlsbergensis and mitochondrial DNA (mtDNA) purified from C. gloeosporioides total DNA as probes. The average size of the rDNA repeat unit of the isolates was 9·2 kb, although there was considerable size variation. Isolates obtained from different hosts never had the same rDNA or mtDNA RFLP patterns. With the exception of those from mango, isolates could not easily be grouped but could be distinguished in relation to their host source within geographical localities. There was no RFLP that could be used diagnostically on a worldwide basis to identify isolates from avocado, banana or papaya. In contrast, isolates obtained from mango fruits in the eastern and western hemisphere had the same rDNA and very similar mtDNA restriction fragment-banding patterns.

Physical and genetic mapping of the telomeric major histocompatibility complex region in man and relevance to the primary hemochromatosis gene ( HFE)

We performed pulsed-field gel electrophoresis (PFGE) on genomic DNA from a radiation hybrid (RH) cell line and constructed a high-resolution physical map of the major histocompatibility complex class I region in 6p21.3, where the gene for primary hemochromatosis (HFE) is believed to be located. Due to the intact microsegment of hemizygous human genomic DNA preserved in the RH cell line, simplified and distinct restriction fragment banding patterns were generated. Using the RH cell line, we were able to extend the physical map of the HLA class I region to about 3000 kb, order the known HLA class I genes from centromere to telomere: HLA-B, -C, -E, (-A, -H, -G), and -F, and orient the HLA-F gene along the chromosome. The proximity of HLA-F to HLA-A was confirmed by linkage and linkage disequilibrium analysis. This study shows that RH cell lines can be useful for constructing long-range physical maps in specific regions of the human genome with PFGE. Physical and genetic mapping studies of this region are consistent with a localization of the HFE gene proximal or distal to HLA-A.

Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.

Classical and molecular genetic analyses of his-3 mutants of Neurospora crassa: II. Southern blot analyses and molecular mechanisms of mutagenicity

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants

Mouse lymphoma cells of the L5178Y TK +/−-3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK +/− controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority ( 83 95 ) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.

Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids.

Samples taken from throughout the ranges of distribution of lodgepole pine (Pinus contorta Dougl. ex. Loud.) and jack pine (Pinus banksiana Lamb.) were assayed for Sal I and Sst I chloroplast DNA restriction fragment variation. Although the chloroplast genome is often regarded as highly conserved, at least 2 distinct Sal I and 13 distinct Sst I restriction fragment banding patterns occur in these closely related species. None of the chloroplast DNA restriction fragment banding patterns observed in allopatric lodgepole pine was observed in allopatric populations of jack pine, and vice versa, even though the two species share an extensive zone of sympatry, and gene flow between the species has been reported for nuclear genes. However, several atypical Sst I restriction fragment banding patterns occur only in or near the zone of sympatry. Chloroplasts have been reported to be inherited maternally in the great majority of species studied; however, restriction fragment analyses indicated that chloroplasts are inherited paternally in controlled matings between lodgepole pine (female) and jack pine (male).

Restriction pattern analysis of genomic DNA ofFrankia isolates

Total genomic DNAs ofFrankia isolates were subjected to restriction enzyme digestion and subsequent agarose gel electrophoresis. Restriction fragment banding patterns were unique for each isolate and may therefore be used as a method to distinguish between isolates which may be morphologically indistinguishable. This method might be useful for practical purposes such as tracing specificFrankia strains during field studies.