Variation In Electrophoretic Mobility Research Articles

Levels and Modifications of Both Lens Fiber Cell Connexins Are Affected in Connexin Mutant Mice.

In the lens, cell homeostasis and transparency are supported by intercellular communication facilitated by the channels formed of connexin46 (Cx46) and connexin50 (Cx50). Mutations of these connexins are linked to inherited cataracts. We studied the levels and the variations in electrophoretic mobilities of the immunoreactive Cx46 and Cx50 bands between 1 and 21 days after birth in the lenses of wild-type mice and homozygous animals from two different mouse models of connexin-linked cataracts (Cx46fs380 and Cx50D47A). In Cx50D47A mice, the expression of the mutant Cx50 reduced the normal phosphorylation of the co-expressed wild-type Cx46. In both models, levels of the mutant connexin and the co-expressed wild-type connexin decayed more rapidly than in wild-type mice but with different time courses. In the Cx46fs380 mice, modeling suggested that Cx50 degradation could be explained by the mixing of mutant Cx46 with wild-type Cx50. However, in Cx50D47A mice, similar modeling suggested that mixing alone could not explain the decrease in Cx46 levels. These data highlight the complex influences between two connexin proteins expressed in the same cell, some of which occur through direct mixing, while others occur indirectly, as in Cx50D47A mice, where the expression of the mutant connexin causes endoplasmic reticulum stress and impaired differentiation.

Quantification of the bisphosphonate alendronate using capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing.

A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8-2000ng/mL and an LOD of 2ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.

Evaluation of protein separation mechanism and pore size distribution in colloidal self-assembled nanoparticle sieves for on-chip protein sizing.

The separation behavior of 6.5-66 kDa proteins in silica particle array-based sieves formed by colloidal self-assembly in microchips is reported across a pore size range of 22-103 nm. The protein separation and resolution improves markedly with decreasing pore size. The variation of electrophoretic mobility with molecular weight of SDS-protein complexes and with particle size was evaluated using the Ogston sieving equation for a random pore gel structure, and using the modified Giddings equation developed by Wirth for uniform pore structures. The Wirth/Giddings equation provides the best fit for estimation of molecular weight of proteins, and demonstrates that even though experimental evidence shows there is some dispersion in measured pore sizes, these structures can best be described as having a uniform pore size.

Monolayers of silver nanoparticles on positively charged polymer microspheres

Abstract The deposition of silver nanoparticles on positively charged polystyrene microspheres was studied by micro-electrophoretic and concentration depletion methods involving UV–vis spectrometry. The influence of ionic strength varied between 10 −4 to 3 × 10 −2 M, was determined. Primarily, the electrophoretic mobility variations of latex upon the addition of controlled amount of silver nanoparticles were measured. These dependencies were quantitatively interpreted in terms of the 3D electrokinetic model previously used for planar interface. This allowed to determine the coverage of nanoparticles on microspheres under in situ conditions for incomplete monolayer range. Additionally, the maximum coverage of silver nanoparticles was precisely determined by measuring the absorption spectrum of the silver suspension acquired by ultrafiltration after the deposition step. It was shown that maximum coverage monotonically increased with ionic strength attaining 0.52 for the ionic strength of 3 × 10 −2 M. This effect, attributed to the decreasing range of lateral electrostatic interactions among deposited particles, was accounted for by the random sequential adsorption model. The obtained results proved the feasibility of preparing silver nanoparticle shells on polymeric carrier microspheres having well-defined coverage and structure. Also, our measurements confirmed the validity of the 3D electrokinetic method for a quantitative interpretation of nanoparticle deposition on microspheres.

Electrophoretic mobility confirms reassortment bias among geographic isolates of segmented RNA phages

BackgroundSex presents evolutionary costs and benefits, leading to the expectation that the amount of genetic exchange should vary in conditions with contrasting cost-benefit equations. Like eukaryotes, viruses also engage in sex, but the rate of genetic exchange is often assumed to be a relatively invariant property of a particular virus. However, the rates of genetic exchange can vary within one type of virus according to geography, as highlighted by phylogeographic studies of cystoviruses. Here we merge environmental microbiology with experimental evolution to examine sex in a diverse set of cystoviruses, consisting of the bacteriophage ϕ6 and its relatives. To quantify reassortment we manipulated – by experimental evolution – electrophoretic mobility of intact virus particles for use as a phenotypic marker to estimate genetic exchange.ResultsWe generated descendants of ϕ6 that exhibited fast and slow mobility during gel electrophoresis. We identified mutations associated with slow and fast phenotypes using whole genome sequencing and used crosses to establish the production of hybrids of intermediate mobility. We documented natural variation in electrophoretic mobility among environmental isolates of cystoviruses and used crosses against a common fast mobility ϕ6 strain to monitor the production of hybrids with intermediate mobility, thus estimating the amount of genetic exchange. Cystoviruses from different geographic locations have very different reassortment rates when measured against ϕ6, with viruses isolated from California showing higher reassortment rates than those from the Northeastern US.ConclusionsThe results confirm that cystoviruses from different geographic locations have remarkably different reassortment rates –despite similar genome structure and replication mechanisms– and that these differences are in large part due to sexual reproduction. This suggests that particular viruses may indeed exhibit diverse sexual behavior, but wide geographic sampling, across varying environmental conditions may be necessary to characterize the full repertoire. Variation in reassortment rates can assist in the delineation of viral populations and is likely to provide insight into important viral evolutionary dynamics including the rate of coinfection, virulence, and host range shifts. Electrophoretic mobility may be an indicator of important determinants of fitness and the techniques herein can be applied to the study of other viruses.

Salt Modulates Bacterial Hydrophobicity and Charge Properties Influencing Adhesion of Pseudomonas aeruginosa (PA01) in Aqueous Suspensions

The influence on cell hydrophobicity of differential extension with ionic strength of lipopolysaccharide molecules (LPS), which exist as charged and uncharged polymers at the surface of the gram-negative bacterium Pseudomonas aeruginosa (PA01), has been investigated. Attenuated total reflection infrared (ATR-IR) spectral absorptions from a single layer of cells adsorbed to ZnSe increased in intensity with increasing NaCl concentration up to 0.1 mol L(-1). Dynamic contact angle measurements (Wilhelmy plate tensiometry) made with a ZnSe plate having an adsorbed cell layer and the adherence of the cells to hexadecane suggest that PA01 cells were most hydrophobic in contact with 0.1 mol L(-1) NaCl solutions. These data indicate a charge screening induced compression of the charged LPS polymers decreasing the cell-surface approach distance and increasing the cell hydrophobicity due to the greater surface predominance of the uncharged LPS polymers. Interestingly, adsorbed cell layers in 0.3 mol L(-1) NaCl had a lower IR absorption intensity, and PA01 cells suspended in 0.3 mol L(-1) were found to be more hydrophilic, indicating that other factors influence the cell-surface approach distance and hydrophobicity. The examination of cell electrophoretic mobility variation with NaCl concentration suggests that the compression of charged polysaccharides increases the polysaccharide charge density and may also reduce the flow of liquid through the polysaccharide layer affecting the effective potential at the interface, the cell hydrophobicity, and the cell-surface approach distance.

Thermodynamic characterization of calcium-milk protein interaction by isothermal titration calorimetry

In milk, casein micelles are the natural vectors of calcium (Ca) and constitute an ideal vehicle for the delivery of additional Ca, through Ca-protein interactions. Isothermal titration calorimetry (ITC) is a technique of choice in the study of biomolecular interactions and especially in mineral-protein interactions. Calcium-milk protein interactions were thermodynamically characterized by titration of cow’s milk protein with calcium chloride (CC) using ITC. Experiments similar to that of ITC were reproduced and analyzed with other techniques (granulometry and electrophoretic mobility measurement) to better understand the nature of interactions. Binding titration curves were fitted with the “one set of sites model” to determine the thermodynamic parameters (N, 6 mol Ca·mol−1 of cow’s milk protein; K, 1070 mol·L−1; ΔH, 1910 cal·mol−1 of CC; and ΔS, cal·mol−1·K−1). Milk proteins bound up to 8 mg Ca·g−1 of protein at saturation. The global thermodynamic signal obtained upon titration was endothermic. The electrophoretic mobility variations of casein micelles occurring upon CC titration were indicative of Ca-protein interactions of electrostatic nature. Hence, the usual exothermic signal involved in electrostatic interactions was completely hidden by the strong endothermic signal coming from the release of water molecules, either from the hydration shell of the Ca ions and/or dehydration of hydrophobic core of proteins. Particle size variations were indicative of casein micelle retraction upon CC titration.

A Study of Lactate Dehydrogenase (LDH) Isoenzyme is a Biochemical Tumour Marker in Cervical Carcinoma Patients

To establish Lactate dehydrogenase (LDH) isoenzyme is a biochemical tumor marker in cervical carcinoma patients for diagnosis and treatment monitoring of the disease. The Serum and cervical tissue LDH isoenzyme was analyzed qualitatively by Poly Acrylamide Disc Gel Electrophoresis method. The total LDH activity was estimated quantitatively by the method of UV spectrophotometry. 50 untreated cervical carcinoma patients were taken up and compared with age matched healthy females (controls). Out of 50 patients, 5 patients were histologically classified as well differentiated squamous cell carcinoma (Grade-I), 23 were moderately differentiated squamous cell carcinoma (Grade-II) and 22 were poorly differentiated squamous cell carcinoma (Grade-III). Grade-I patients did not show any change, in the serum and cervical tissue LDH isoenzyme fractions compared to the controls but variation in electrophoretic mobility have been observed. Grade-II patients showed two new additional isoenzyme fractions in LDH 2 and LDH 3 locations, where as Grade-III patients showed only three LDH isoenzyme fractions. The total LDH activity in both serum and cervix tissue of normal individuals are 0.458±0.0796 units/ml; and 0.680±0.0218 units/mg. Grade-I patients did not show any change in total LDH activity (serum: 0.384±0.0404 units/ml; cervical tissue: 0.589±0.0292 units/mg; P<0.01). Grade-III patients showed much lower values (serum: 0.284±0.0109 units/ml; cervical tissue: 0.327±0.043 units/mg; P<0.001), where as Grade-II patients showed significant increase in total LDH activity (serum: 0.878±0.0531 units/ml; cervical tissue: 1.296±0.0813 units/mg; P<0.001) respectively. The results suggest that LDH isoenzyme is useful biochemical tumor marker for diagnosis and to assess the grade of malignancy.

Anomalous low-frequency electro-optic behavior of ferric oxide particles in the presence of poly(ethylene oxide)

Electro-optic techniques were used to investigate the influence of poly(ethylene oxide) (PEO) on the surface electric state of positively charged oxide particles. The variations in particle electrophoretic mobility of β-FeOOH particles in the presence of PEO indicate significant changes in the surface electric state of the particles in the concentration interval of PEO 10 −2–10 −1 g dm −3. The electro-optic results for the same conditions were unexpected: no significant difference is observed in the value and the relaxation frequency of particle electric polarizability in the frequency domain of the α-relaxation (detected in the kilohertz range); particle rotational relaxation time also remains unchanged; considerable changes are detected only in the relaxation interval of particle rotation (detected in the hertz range). The obtained results reject the possibility of the formation on the particle surface of a thick polymer layer. A thin adsorption layer cannot explain the significant decrease in particle electrophoretic mobility. The variations in electrophoretic mobility are well correlated with the effects in the domain of particle rotation. A possible explanation of the observed effects is proposed, based on our previous investigations of the effects in the low-frequency domain. The presented results demonstrate that the important information on the electrokinetic charge distribution is not found in the domain of the α-dispersion, but in the domain of particle rotation.

The alteration of cell membrane charge after cultured on polymer membranes

In this work, cell electrophoresis, measuring the electrophoretic mobility of cells, was used to investigate the variation of surface charge property of cells after cultured on different polymer membranes. HepG2 cell line, derived from a well-differentiated, human hepatoma, was used as a model cell. The polymer biomaterials used in this study included polyvinyl alcohol (PVA), poly(ethylene- co-vinyl alcohol) (EVAL), and polyvinylidene fluoride (PVDF). For cells cultured in the presence of serum, cell mobility after being cultured on PVA substrates was considerably higher than that on EVAL or PVDF substrates. This effect was completely suppressed by cycloheximide (CHX) in the serum-free medium. Taken together, the cell surface charge property can be altered after cells cultured on different polymer substrates. The precise mechanism by which the variation of electrophoretic mobility of cultured cells is unknown, but it is reasonable to assume that the polymer substrates could influence the absorption of serum proteins on cell membrane surface to change cell electrophoretic mobility and, simultaneously, to regulate adhesion, growth and function of cultured cells.

Investigating the potential of erythromycin and derivatives as chiral selector in capillary electrophoresis

Macrocyclic antibiotics are present in an increasing number of chiral separation applications. In this study, erythromycin and some derivatives, belonging to the group of macrolide antibiotics, were investigated for their potential as chiral selector. Erythromycin A itself and five related substances namely erythromycin A N-oxide, anhydroerythromycin A, anhydroerythromycin A N-oxide, erythralosamine and erythralosamine N-oxide were included. Twenty-one chiral compounds with a wide difference in physico-chemical properties were used to test the chiral activity of the erythromycins. The chiral compounds were analysed using capillary zone electrophoresis with the erythromycins dissolved in the running buffer at different concentrations ranging from 0.1 to 10mM, with three different BGEs: sodium phosphate pHs 3.0 and 7.0 and sodium borate pH 9.2. The erythromycins showed more interactions with the acidic compounds (as observed with leucovorin, dopa, carbidopa, ketoprofen, indoprofen and warfarin) than with the neutral or weakly basic ones, especially in acidic medium. Unfortunately, no chiral separations were obtained for any of the 21 racemic mixtures. The complexation constants were calculated for the compounds showing interaction, based on the variation of electrophoretic mobility of the compounds in the presence of different concentrations of erythromycins. Finally, the size of erythromycin A was calculated using computational modelling. It was shown that the aglycone ring is only half as big as the β-cyclodextrin cavity, giving a possible explanation for the negative response of erythromycin in this study.

Change in electrophoretic mobility of PC12 cells after culturing on PVA membranes modified with different diamines

The cell-biomaterial interaction is of extreme importance in regulating the numerous functions necessary for cell adhesion, growth, and differentiation. In the current study, electrophoresis was used to investigate the interactions between cells and biomaterials by measuring the change in electrophoretic mobility of pheochromocytoma (PC12) cells after they were cultured on the poly (vinyl alcohol) membranes modified with different diamines. Variations in cellular activity and electrophoretic mobility of cultured cells were compared. It was found that the intracellular metabolism and the cell surface charge properties were altered after cells contacting biomaterials and the variation of the latter occurred earlier than that of the former. Although the precise mechanism by which the variation of electrophoretic mobility of cultured PC12 cells was unknown, the biomaterials could influence the cell mobility within a short incubation time. It was hypothesized that changes in extracellular matrix components of cell surface may be in part responsible.

Induction of glycosylation in human C-reactive protein under different pathological conditions.

As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 microg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein-protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.

Pressure pulse injection: a powerful alternative to electrokinetic sample loading in electrophoresis microchips.

We present a new and simple capillary electrophoresis microsystem in which the sample is injected hydrodynamically using a pressure pulse. This technique maintains the sample composition, in contrast to a classical electrokinetic injection, in which the magnitude of the electric field in the sample reservoir in combination with variations in electrophoretic mobility can lead to a biased injection. The sample is loaded using a well controlled and variable pressure pulse (0.1-1.0 s) generated by the mechanical actuation of a flexible membrane placed on the chip sample reservoir. A fluorescein/calcein-containing borate-Tris-hydroxymethylaminoethane (TRIS) sample solution is taken as a model system for CE analysis. The separation results using pressure pulse injection clearly demonstrate the advantages of our technique. In addition to the reduced bias due to the absence of an electrode in the sample well, this method allows injection of variable plug volumes by simply changing the pulse length. Moreover, a very high speed, repeatability, and sensitivity of the separation is obtained.

Electrophoresis of a Concentrated Dispersion of Nonrigid Particles

Electrophoresis is one of the most important analytical tools for the quantification of charged entities in a dispersing medium. Although the electrophoresis of rigid entities has been studied extensively in the literature, corresponding analysis on nonrigid entities is relatively limited. The present study focuses on the electrophoresis of a concentrated dispersion of nonrigid particles for the case when the effect of double layer polarization may be significant. In particular, the effects of the surface potential of the particle, the volume fraction of the particles, the ratio (viscosity of the medium inside the particle/viscosity of the medium outside the particle), and the double layer thickness are discussed. Various phenomena specific to nonrigid entities that are not observed for the case of rigid entities are observed in this study, which include, for example, the fact that the variation of electrophoretic mobility as a function of double layer thickness exhibits an inflection point. We show that, under the same conditions, the electrophoretic mobility of a nonrigid particle is larger than that of a rigid particle, and this is pronounced if the double layer is thin.

Characterization and immunogenicity of the CagF protein of the cag pathogenicity island of Helicobacter pylori.

Helicobacter pylori infection causes severe gastroduodenal diseases in humans. Its virulence is strongly increased by the presence of the cag pathogenicity island (cag PAI). It has been shown that CagA, a major antigen in humans, is translocated to the host cell via a secretion system encoded by the cag PAI. The roles of many of the proteins encoded within the cag PAI are not known. Here we report on the cloning and expression of CagF, one of those proteins. We show that CagF is associated to the outer membrane of H. pylori G27 and that the protein is always expressed with electrophoretic mobility variations among the 20 strains tested here. We have also found that natural infection with H. pylori is able to induce antibodies against CagF.

Rhizobium mongolense sp. nov. is one of three rhizobial genotypes identified which nodulate and form nitrogen-fixing symbioses with Medicago ruthenica [(L.) Ledebour

Medicago ruthenica [(L.) Ledebour] is native to inner Mongolia where rhizosphere samples were collected for the isolation of 106 rhizobial cultures. Besides nodulating the original trap host, the isolates formed nitrogen-fixing symbioses with Phaseolus vulgaris. Only half of the isolates nodulated alfalfa (Medicago sativa), but these did not form nitrogen-fixing symbioses. Rhizobium tropici also formed nitrogen-fixing symbioses with Medicago ruthenica. A total of 56 distinctive multilocus electrophoretic types (ETs) were identified among 94 of the 106 isolates which were analysed for variation in electrophoretic mobility of 12 enzyme loci. One isolate (USDA 1920) possessed a unique ET, while the ETs of the other isolates formed two weakly divergent subgroups approximately equal in size. It was concluded from small subunit rRNA gene sequences of eight isolates of Medicago ruthenica that they belonged to the genus Rhizobium and not to the genus Sinorhizobium which is more commonly associated with Medicago. Genomic similarity, determined from DNA hybridization analysis, between USDA 1920 and the strain representing the remaining isolates (USDA 1844) was lower than 20%. Based upon these observations it was concluded that at least three genomic species of rhizobia form nitrogen-fixing symbioses with Medicago ruthenica. One of these genomic species is R. tropici, another is represented by the single isolate USDA 1920 and the name Rhizobium mongolense is proposed for the third genomic species represented by USDA 1844.

Comparison of DNA bending by Fos-Jun and phased A tracts by multifactorial phasing analysis.

Studies of DNA bending by Fos and Jun using different methods have yielded contradictory results. Whereas gel electrophoretic phasing analysis indicates that Fos and Jun bend DNA, results obtained through X-ray crystallography and ligase-catalyzed cyclization suggest that they do not. To test the assumptions underlying phasing analysis and to examine DNA bending by Fos and Jun, a multifactorial phasing analysis approach based on the distinct electrophoretic mobilities of DNA fragments of diverse shapes was developed. In this approach, the spacing between the bends, the length of sequences flanking the bends, and the acrylamide concentration in the gel are varied. Two closely spaced intrinsic bends with long flanking sequences had the same effect on electrophoretic mobility as a single bend corresponding to the sum of the bends when they were arranged in phase, and the difference between the bends when they were arranged out of phase. Based on the phase-dependent electrophoretic mobility variation of fragments containing intrinsic DNA bends of different magnitudes, three criteria for determination whether the phase-dependent mobility variation of protein-DNA complexes is caused by DNA bending were adopted. Complexes formed by the bZIP domains of Fos and Jun fulfilled each of these criteria. First, the electrophoretic mobility variation induced by Fos and Jun was proportional to that caused by an intrinsic bend over a broad range of acrylamide concentrations. Second, the mobility difference between fragments containing in phase and out of phase bends was reduced by an increase in the separation between the bends. The separation between the bends had the same effect on the electrophoretic mobility variation caused by Fos and Jun as well as intrinsic bends on long DNA fragments at low acrylamide concentrations. Third, on short DNA fragments analyzed at high acrylamide concentrations, two intrinsic bends separated by long spacers caused a larger decrease in electrophoretic mobility when they were out of phase than when they were in phase. This reversal of the phase dependence of the electrophoretic mobility variation was also observed for complexes formed by truncated Fos and Jun. Thus, the phase-dependent mobility variation of Fos and Jun complexes is due to DNA bending.

Migration behavior of dichlorophenols for replicate separations without replenishment of buffer electrolyte in micellar electrokinetic capillary chromatography

The migration behavior of dichlorophenols in replicate separations, without replenishment of buffer electrolyte between runs, in micellar electrokinetic capillary chromatography (MECC) was investigated. The results indicated that the pH of the buffer electrolyte drifts as a result of electrolysis of the buffer solution during electrophoresis and that the pH drift, being independent of the micelle concentration, is responsible for the variation in electrophoretic mobilities of analytes in MECC when the buffer is not replenished. The influences of the buffer pH and micelle concentration on the migration behavior of analytes were demonstrated to be correlated at small micelle concentrations. The variation of electrophoretic mobility of an analyte as a function of buffer pH at a given micelle concentration is quantitatively described. 2,6-Dichlorophenol is selected for illustration. The variation of electrophoretic mobility agrees satisfactorily between predicted and observed data.

The complete karyotype of Aspergillus niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies.

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, aglA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant strains for the purpose of chromosome assignment.