Fluorescent Primers Research Articles

Acute Myeloid Leukemia with Normal Karyotype: Analysis of FLT3, NPM1, MLL and CEBPA Gene Abnormalities.

Acute myeloid leukemia with normal karyotype (AML-NK) is markedly heterogeneous depending on the presence or absence of certain genetic alterations. We studied the prevalence of different abnormalities, namely of the FLT3, NPM1, CEBPA and MLL genes, in AML-NK patients and tried to correlate them with clinicopathological findings. Techniques used were as follows: FLT3-ITD (internal tandem duplications), PCR using fluorescent primers, analysis in automatic sequencer; FLT3-TKD (D385), PCR, fragment cut with EcoRV restriction enzyme run in agarose gel; NPM1, RT-PCR and automatic sequencing of a fragment containing exon 12; CEBPA, PCR and automatic sequencing of the entire coding region; MLL-PTD, RT-PCR with analysis in agarose gel. The following data from patients were recorded: sex, age, FAB classification, WBC and platelet count, hemoglobin and LDH level, response to induction, survival time and cause of death. The incidence of FLT3 alterations (all ITDs) was 10/23 (43.5%), varying from 1 to 10 ITDs; NPM1 was mutated in 12/24 cases (50.0%) and MLL-PTD in 2/24 patients (8.3%); for CEBPA, of the 10 cases in which all 3 amplicons (1A, 2B, 3C) were analysed, 6 had a mutation mostly in amplicon 2B (5 cases). More than one genetic abnormality was present in 9 cases: six showed coexistence of FLT3-ITD and NPM1 mutation, one of MLL and CEBPA mutations, one of FLT3-ITD and CEBPA mutation and one of FLT3-ITD, NPM1 and CEBPA. Regardind correlation with clinical characteristics, we observed that 72.7% of nucleophosmin mutated (NPM1+) cases were CD34 negative and 91.7% of CD34+ patients were NPM1 negative (p=0.003). There is a tendency, although not significant, for CEBPA+ cases to correlate with low WBC counts and for patients with high WBC count to be NPM1+ and FLT3-ITD+ (odds ratio of 3.0 and 3.33, respectively). All NPM1+ patients treated with appropriate induction therapy achieved complete remission; 22.2% of NPM1 negative patients were refractory to therapy. No significant correlation was observed between survival and presence or absence of any of the genetic alterations. This work was funded by an unrestricted grant from Roche Farmaceutica - Portugal.

Population Genetic Structure in Apricot ( Prunus armeniaca L.) Cultivars Revealed by Fluorescent-AFLP Markers in Southern Xinjiang, China

Population-wide genetic structure was studied using fluorescent-AFLP markers on 85 apricot ( Prunus armeniaca L.) cultivars collected from Kuche, Kashi, Hetian in the Tarim Basin, southern Xinjiang Uygur Autonomous Region of China. The purpose of this study was to determine the genetic structure and genotypic diversity among the different eco-geographical populations. Based on the results from this study, 8 pairs of fluorescent-AFLP primers showed clear electrophoregram and high polymorphism amongst the 64 pairs of EcoR|/ Mse|( Mse|- a FAM fluorescent marked primer) primers screened. There was a significant polymorphic difference for the same primer pair in different populations and for the same population with different primer pairs. The percentage of polymorphic loci ( P) at species level was higher than Kuche, Hetian, Kashi population levels, respectively. The Nei's gene diversity index ( H) and Shannon's information index ( I) at species level were higher than those of Kuche, Hetian, and Kashi at population level, respectively. H and I of Kuche population were the highest amongst the three populations. Apricot population genetic diversity was found mainly within the population. Genetic differentiation coefficient between populations ( G ST ) was 0.0882. Gene flow Nm between the populations was 5.1689. Population genetic identity was between 0.9772–0.9811 and genetic distance was between 0.0191–0.0232. These results further indicated that the similarity between populations was higher and the genetic distance between populations was smaller. The UPGMA cluster analysis indicates that the geographical populations at Kuche, Kashi, Hetian were relatively independent Mendelian populations. Concurrently, there was also partial gene exchange between the populations. All the evidences indicated that the genetic diversity in Kuche population was the highest, suggesting that it could be a transition population from wild apricot to cultivated apricot. There were abundant genetic diversities in apricot cultivar populations in southern Xinjiang, China, which provide promising germplasm for further breeding and theoretical basis for biodiversity conservation and utilization for apricot population in this area.

Abstract 2378: The MinK G38S Polymorphism Associates with Atrial Arrhythmias after Subarachnoid Hemorrhage

Background: Subarachnoid hemorrhage (SAH) leads to significant death and disability. Cardiac abnormalities after SAH include atrial arrhythmias (AAs). The MinK (KCNE1) G38S polymorphism has been associated with atrial fibrillation, with the G38 allele being linked to increased action potential duration in heterologous expression systems. The relationship of AAs and ion channel polymorphisms after SAH is not known. Methods: In 148 patients admitted for acute, aneurysmal SAH, EKG parameters, 24-hour Holter monitors, serum troponin (TnI), potassium (K), magnesium (Mg), and echocardiograms (ejection fraction [EF] and left atrial diameter [LA D]) were examined. MinK G38S was genotyped using fluorescent primers and analyzed with an ABI Prism 7000. Results: No patient had AAs on presentation, but AAs developed in 101 (68%) patients: SVT (95; 64%); fibrillation (9; 6%); flutter (4; 3%); idioventricular rhythm (2; 1%); and 3° block (1; 1%). AAs were not associated with PR or QTc intervals, TnI, K, Mg, EF, LA D, or stroke severity. AAs were associated with older age (58±9 vs 51±10yrs; p<0.001), lower heart rate (HR; 80±10 vs 84±14bpm; p=0.02), and higher HR variability (SDNN; 97±34 vs 85±32; p=0.05). The MinK S38 allele was associated with AAs (Figure ; GS & SS 70% vs GG 51%; p=0.02), but not LA D. Conclusions: Atrial arrhythmias are common following SAH and the MinK S38 allele is associated with an increased frequency of atrial arrhythmias. The SAH-induced catecholamine surge, followed by increased parasympathetic activity, may enhance the shortening of the atrial effective refractory period in patients carrying the MinK S38 allele and increase susceptibility to atrial arrhythmias.

Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein-RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.

The identification, diversity and prevalence of trypanosomes in field caught tsetse in Tanzania using ITS-1 primers and fluorescent fragment length barcoding

We report on the development of two generic, PCR-based methods, which replace the multiple species-specific PCR tests used previously to identify the trypanosome species carried by individual tsetse flies. The first method is based on interspecies size variation in the PCR product of the ITS-1 region of the ribosomal RNA (rRNA) locus. In the second approach, length variation of multiple fragments within the 18S and 28S rRNA genes is assayed by PCR amplification with fluorescent primers; products are subsequently sized accurately and rapidly by the use of an automated DNA sequencer. Both methods were used to identify samples collected during large-scale field studies of trypanosome-infected tsetse in Tanzania in the National Parks of Tarangire and Serengeti, and the coastal forest reserve of Msubugwe. The fluctuations of trypanosome prevalence over time and two different field seasons are discussed. As well as facilitating the identification of trypanosome species with increased speed, precision and sensitivity, these generic systems have enabled us to identify two new species of trypanosome.

Group A rotavirus genotypes in Germany during 2005/2006

During the 2005/2006 winter season a total of 802 group A positive rotavirus specimens of patients from different regions throughout Germany were genotyped. Amplicons from a one-tube RT-PCR were typed by analysis of their (type-specific) size using type-specific primers, fluorescent consensus primers and a capillary sequencer for detection. While G1P[8] was predominant (45.8%), G9P[8] has emerged as the second most frequent genotype combination (37.7%). The distribution of genotypes was heterogeneous, regional frequencies regarding G1 and G9 were ranging from 15.0 to 89.3% and from 7.1 to 67.7%, respectively. Furthermore, a few human rotavirus G10P[6] and G10P[8] infections were observed.

Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection

This protocol describes capillary array electrophoresis single-strand conformation polymorphism (CAE-SSCP), a screening method for detection of unknown and previously identified mutations. The method detects 98% of mutations in a sample material and can be applied to any organism where the goal is to determine genetic variation. This protocol describes how to screen for mutations in 192 singleplex or up to 768 multiplex samples over 3 days. The protocol is based on the principle of sequence-specific mobility of single-stranded DNA in a native polymer, and covers all stages in the procedure, from initial DNA purification to final CAE-SSCP data analysis, as follows: DNA is purified, followed by PCR amplification using fluorescent primers. After PCR amplification, double-stranded DNA is heat-denatured to separate the strands and subsequently cooled on ice to avoid reannealing. Finally, samples are analyzed by capillary electrophoresis and appropriate analysis software.

Analysis of genital Candida albicans infection by rapid microsatellite markers genotyping

Candida albicans (C. albicans) infection, often occurring in genital candidiasis, has increased dramatically recently. Developing an efficient C. albicans typing method may contribute to understanding its epidemiological characteristics and guiding efficient treatment. We used rapid microsatellite genotyping assay for interstrain differentiation of C. albicans isolates and explored some characteristics of its spread. DNA was extracted from C. albicans isolates from gentalia, recta and mouths of 39 female cases and 27 male cases of genital candidiasis. Three fluorescent primers for the microsatellite markers in conserved genes (CDC3, EF3 and HIS3) of C. albicans were used to amplify the isolates DNA by PCR. Fluorescent signals were read with an automatic sequencer and analyzed with GeneScan software. Analysis of the three microsatellites markers showed 18 gene allelic associations in genital C. albicans infected patients: 10 allelic associations in female and 11 allelic associations in male, of which 3 allelic associations shared by both genders covered 71% of infections. The most dominant allele association of pathogenic strains for both genders was 116:124, 122:131, 160:200 that covered about 50% of infection. Gentalia and recta shared the same strains in 80% of female patients, but in only 3.8% of male patients. There were 2.7% female patients, but no males, with same strain in both gentalia and mouths. Five of seven genital C. albicans infected couples had the same allelic associations of which 4 were the dominant pathogenic C. albicans susceptible for both genders. The predominant allelic association of the pathogenic strain in genital C. albicans infection is 116:124, 122:131, 160:200. Vaginal pathogenic strains are probably maintained from the rectal reservoir. Pathogenic strains of male patients are probably from frequent sexual intercourse. The aggressiveness of some strains varies with gender.

Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression

Fibulin-2, an extracellular matrix protein expressed by normal epithelia, was found to be down-regulated in several breast cancer cell lines. Fibulin-2 protein expression was also decreased in breast cancer tissue samples as evaluated by immunohistochemistry. Reintroduction of Fibulin-2 into breast cancer cell lines that do not express Fibulin-2 reduced cancer cell motility and invasion in vitro but had no effect on cell growth and adhesion properties. Together with evidence that Fibulin-2 contributes to wound healing and inhibits smooth muscle cell migration, our findings suggest that loss of Fibulin-2 expression may facilitate migration and invasion in breast cancer.

SNP‐SCALE: SNP scoring by colour and length exclusion

AbstractHere we present SNP‐SCALE, a new single nucleotide polymorphism genotyping protocol based on allele‐specific polymerase chain reaction (AS‐PCR), but with two critical modifications designed to maximize accuracy, efficiency and throughput and minimize costs. The first is the addition of a locked nucleic acid (LNA) at the 3′‐SNP position of each of the two allele‐specific forward primers. The second is the addition of size‐differentiating M13 tails at the 5′ end of each primer, which facilitates the use of universal fluorescent primers. The use of LNAs makes SNP‐SCALE significantly more sensitive and specific than standard AS‐PCR. Moreover, SNP‐SCALE is ideally suited to a touchdown‐like PCR protocol, such that PCRs can be standardized, PCR optimization is straightforward, and medium‐high throughput can be readily achieved. We anticipate that the SNP‐SCALE protocol will be useful to molecular ecologists for a broad range of studies.

Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here, we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods and revealed by ordination were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP, and this may reduce the sensitivity to changes in non-dominant populations within the community. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. T-RFs should therefore be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to equipment required to perform fluorescent T-RFLP.

Resolution of ambiguous low‐level positive quantitative polymerase chain reaction results in TEL‐AML1 positive ALL using a post‐PCR fluorescent oligoligation method*

The interpretation of low-level or non-reproducible amplification results in clinical quantitative polymerase chain reaction (Q-PCR) assays can be difficult to definitively resolve. Concerning minimal residual disease detection in leukaemia, indeterminate low-level results might create prognostic or therapeutic dilemmas. We evaluated low-level, ambiguous Q-PCR results in a study of paired diagnostic and end-induction (day 29) TEL-AML1 positive acute lymphoblastic leukaemia samples utilising a novel fluorescent primer ligation detection assay. The data presented here indicate that a significant number of low-level apparent Q-PCR positive results may be spurious or non-specific in nature, requiring additional technical manoeuvres for confirmation of true positive cases.

Relationship between polymorphisms 804C/A and 252A/G of lymphotoxin- α gene and −308G/A of tumor necrosis factor α gene and diabetic retinopathy in Japanese patients with type 2 diabetes mellitus

To clarify whether polymorphisms of the lymphotoxin- α (LTA) gene and tumor necrosis factor α (TNF- α) gene were related to diabetic retinopathy (DR), we performed a case-control study in 251 Japanese patients with type 2 diabetes mellitus participating in a multicenter research protocol. Genetic analyses were performed by using a fluorescent allele-specific DNA primer assay system. Diabetic retinopathy was diagnosed in a masked manner by an independent ophthalmologist using fundus photographs and was classified as nondiabetic retinopathy (NDR), nonproliferative retinopathy (NPDR), and proliferative retinopathy (PDR). The results showed that the genotype frequencies of 804C/A in exon 3 and 252A/G in intron 1 of the LTA gene were not significantly different among patients with NDR, NPDR, and PDR. A allelic frequency of the TNF- α gene (−302A/G in promoter) was also identical among NDR, NPDR, and PDR groups. Multivariate logistic regression analyses showed that significant associations with DR were glycosylated hemoglobin level and diabetes duration, but not polymorphisms of the LTA gene or TNF- α gene. In conclusion, the present study showed no association between polymorphisms 804C/A and 252A/G of the LTA gene and −302A/G of the TNF- α gene and DR in Japanese type 2 diabetic patients.

In vivo expression of the heat stable ( estA) and heat labile (e ltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.

An accurate fluorescent assay for quantifying the extent of RNA editing.

Recent data suggest that small differences in editing efficiency can have significant functional consequences. Here we present a fluorescent poisoned primer extension assay that is capable of distinguishing editing efficiency differences as low as 5%. For a poison-primer extension assay to be accurate, the extension product must stop at the intended base. Sometimes, however, it runs beyond. We tested the effect of specific enzyme-terminator combinations on the amount of run through. In the worst cases it accounted for 70% of the total signal, and in the best cases <5%. In addition, the specific base can affect run through, with G producing the least. The accuracy of the assay was demonstrated on templates derived from mixed plasmids and then verified on two biological substrates. Using either a K(+) channel mRNA that contains a site for adenosine deamination or an ndhB mRNA that contains a site for cytidine deamination, the editing efficiency predicted by the assay closely matched that predicted by bulk sequencing of individual cDNA clones. This assay should prove useful for analyzing small changes in editing efficiency or for quantifying single nucleotide polymorphisms.

Systematic Analysis of Arabidopsis Organelles and a Protein Localization Database for Facilitating Fluorescent Tagging of Full-Length Arabidopsis Proteins

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation.

Angiotensinogen gene polymorphism (Met235Thr) influences visceral obesity and insulin resistance in obese Japanese women

To investigate the relationship between angiotensinogen (AGT) Met235Thr polymorphism (M235T) and human obesity, because AGT is regarded as one of the cytokines produced from adipocytes and serum AGT concentrations are reported to be positively correlated with body mass index. One hundred and twenty obese Japanese women (age, 58.8 ± 9.4 years; body mass index, 32.2 ± 4.9 kg/m 2) were enrolled. Angiotensinogen genotypes were determined with a fluorescent allele-specific DNA primer assay system. Subjects were divided into M/M, M/T, and T/T groups. Control subjects comprised 146 healthy age-matched women. Clinical characteristics and the effects of diet and exercise therapy for 6 months were compared among the 3 genotypes. The genotype frequencies of AGT M235T polymorphism were in accordance with the Hardy-Weinberg equation (obese: M/M, 6.7%; M/T, 27.5%; T/T, 65.8%; control: M/M, 6.8%; M/T, 21.2%; T/T, 71.9%). The frequency of the T allele did not differ between obese and control subjects (0.80 vs 0.83). As the number of obese women with M/M genotype was only 8, comparisons of the characteristics and outcomes of weight reduction therapy were performed only between subjects with M/T genotype and T/T genotype. In the T/T group, % body fat and waist circumference at baseline were significantly greater than in the M/T group (36.3% ± 4.8% vs 33.8% ± 4.7%, P = .0105; 107.9 ± 10.9 vs 102.6 ± 7.9 cm, P = .0428, respectively). Before the weight reduction therapy, significantly higher insulin and higher homeostasis model assessment (HOMA-R) were demonstrated in the T/T group than in the M/T group (9.1 ± 5.5 μU/mL vs 5.9 ± 4.4 μU/mL, P = .0056; 2.3 ± 1.4 vs 1.6 ± 1.3, P = .0252, respectively). Both systolic and diastolic blood pressure at baseline in the T/T group tended to be higher than those in the M/T group, but the differences were not significant. No genotype-dependent difference in energy expenditure or outcome of weight reduction therapy was observed with respect to AGT M235T polymorphism. After the diet and exercise therapy, the blood pressure in the T/T group tended to be higher than that in the M/T group, but the difference was not significant. We demonstrated that the T/T genotype of the AGT M235T gene polymorphism was positively related to visceral obesity and hyperinsulinemia in obese Japanese women. Blood pressure did not show genotype-specific differences before or after the treatment. Further studies of the association between obesity and this gene polymorphism should contribute to understanding and treating obesity-related diseases.

Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHASE327Q and seHASE327K were expressed at low levels, whereas seHASE327D and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHASE327K,K48E protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (approximately 0.6 MDa) was made by seHASE327K,K48E and seHASK48E. The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

A NEW HIGH-PERFORMANCE PCR DIAGNOSTIC FOR THE DETECTION OF PYRETHROID KNOCKDOWN RESISTANCE KDR IN ANOPHELES GAMBIAE

Monitoring the spread of the knockdown resistance allele kdr in areas of extensive pyrethroid use is critical to vector-control projects. Currently available methods for detecting kdr from DNA samples are characterized by poor amplification, time-consuming steps, and primers that exhibit frequent null alleles. We describe a new PCR diagnostic that uses fluorescent primers based on conserved priming sites and enables simple detection of the kdr allele on a sequencer. Using samples from a West African Anopheles gambiae population, we show that the new PCR yielded significantly higher rates of amplification and more accurate estimates of kdr frequency. The method works equally well for the leucine to phenylalanine substitution found in West Africa and the East African leucine to serine substitution.