Flat-bed Isoelectric Focusing Research Articles

Isoelectric Focusing on Non-Denaturing Flatbed Gels.

Isoelectric focusing (IEF) serves as a very useful procedure for cell protein separation and characterization. We have used this method to study antibody clonotype changes. Here we discuss the use of a sensitive native flatbed isoelectric focusing method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus, who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera samples collected over several years were used for analysis using flatbed IEF. Following electrofocusing, the gel is analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane to determine oligoclonality of the anti-Ro 60 containing sera.

Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels.

Due to their heterogeneity and huge differences in abundance, the detection and identification of all proteins expressed in eukaryotic cells and tissues is a major challenge in proteome analysis. Currently the most promising approaches are sample prefractionation procedures prior to narrow pH range two-dimensional gel electrophoresis (IPG-Dalt) to reduce the complexity of the sample and to enrich for low abundance proteins. We recently developed a simple, cheap and rapid sample prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated gels. Complex sample mixtures are prefractionated in Sephadex gels containing urea, zwitterionic detergents, dithiothreitol and carrier ampholytes. After IEF, up to ten gel fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of two-dimensional (2-D) gel electrophoresis. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF, since the prefactionated samples are not eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with subsequent IPG-IEF. Prefractionation allows loading of higher protein amounts within the separation range applied to 2-D gels and facilitates the detection of less abundant proteins. Also, this system is highly flexibile, since it allows small scale and large scale runs, and separation of different samples at the same time. In the current study, this technology has been successfully applied for prefractionation of mouse liver proteins prior to narrow pH range IPG-Dalt.

Multiple light-harvesting II polypeptides from maize mesophyll chloroplasts are distinct gene products

The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyllbinding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zea mays LHCII. Each protein is purified to homogeneity, subjected to direct protein sequencing and the sequences compared with those deduced from lhcb genes in maize and other organisms. All of the six proteins are distinct gene products, since they show differences in their primary structure. Three apoproteins are identified as products of type I lhcb genes and one each as type II and type III gene products. A sixth protein does not fit the requirements for any of the lhcb genes so far cloned and is therefore probably the product of an Ihcb gene type not yet described. Our results clearly show that the major source of LHCII protein heterogeneity is the expression of many lhcb genes. Fractionation of maize LHCII by non-denaturing flat-bed isoelectric focusing resolves at least five major isoforms showing distinct differences in their polypeptide composition and also differing in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment-binding properties.

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78–4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.

Cold-Resistant and Cold-Sensitive Maize Lines Differ in the Phosphorylation of the Photosystem II Subunit, CP29.

The effects of low temperature on the relative contributions of the reaction center and the antenna activities to photosystem II (PSII) electron transport were estimated by chlorophyll fluorescence. The inhibition of PSII photochemistry resulted from photo-damage to the reaction center and/or a reduced probability of excitation energy trapping by the reaction center. Although chill treatment did not modify the proportion of the dimeric to monomeric PSII, it destabilized its main light-harvesting complex. Full protection of the reaction center was achieved only in the presence of the phosphorylated PSII subunit, CP29. In a nonphosphorylating genotype the chill treatment led to photoinhibitory damage. The phosphorylation of CP29 modified neither its binding to the PSII core nor its pigment content. Phosphorylated CP29 was isolated by flat-bed isoelectric focusing. Its spectral characteristics indicated a depletion of the chlorophyll spectral forms with the highest excitation transfer efficiency to the reaction center. It is suggested that phosphorylated CP29 performs its regulatory function by an yet undescribed mechanism based on a shift of the equilibrium for the excitation energy toward the antenna.

EFFECT OF A REPEATED BOUT OF ECCENTRIC EXERCISE ON CREATINE KINASE-MM ISOFORMS 369

The increase in creatine kinase (CK) after a damage-inducing exercise is blunted upon performance of a subsequent bout of similar exercise. CK-MM1(muscle isoenzyme), the pure gene product, is initially released and then converted to structurally modified isoforms. MM2 and MM3, before being cleared by the reticuloendothelial system. This study investigated the release and clearance of MM isoforms following two bouts of eccentric exercise. Eight male subjects performed 50 forced lengthening contractions of the forearm flexor muscles six days apart. Blood samples were collected pre-exercise and at hours 2, 6, 24, 48, 72, 96, and 120 post-exercise for each bout. Total CK (TCK) levels were analyzed spectrophotometrically and MM isoforms were separated using flatbed isoelectric focusing. Densitometric quantification of each MM isoform was acquired as a percentage of total MM enzymatic activity and then converted into absolute activity (IU/L). The MM1:MM3 ratio was calculated for each time point and used to indicate clearance. Values <1 indicate clearance is faster than release, >1 indicate release is greater, and = 1 indicate the rates are equal. Post-bout 1, TCK was significantly elevated peaking at 96h (3530±927 IU/L) (p<0.01). Following bout 2 there was no increase in TCK, demonstrating the typical repeated bout effect. MM1 peaked at 72h after bout 1 (993±424 IU/L), but also showed an increase after bout 2 (166±44). The MM1:MM3 ratio was significantly lower after bout 2(0.58±0.12) when compared to bout 1 (1.61±0.35) (p<0.01). Despite no increase in TCK after bout 2, there was still a slight increase in MM1 indicating that some release of CK occurred. Accelerated clearance of CK appears to be the main factor explaining the blunted response of this enzyme following a repeated bout of exercise.

Convergence of amino acid compositions of certain groups of protein aids in their identification on two-dimensional electrophoresis gels.

The amino acid composition (AAC) versus the protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat-bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root-mean-square rule (Rmsd) with measures the difference between the AAC of chosen protein versus those in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained within 1.5-1.0 with m +/- 3kDA and pI +/- 4. Certain groups of multidomain proteins have quasi-unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC-PI method may become an additional search tool for protein functions.

Changes in proteins and peroxidases induced by compatible pollination in the ovary of Nicotiana tabacum L. ahead of the advancing pollen tubes

Proteins and peroxidases produced by the ovules and placenta of tobacco (Nicotiana tabacum L.) in response to compatible pollination were analyzed by two-dimensional polyacrylamide gel electrophoresis and by enzyme staining in flat-bed native isoelectric focusing gels. For two-dimensional gels, ovaries were sampled at 36 h after pollination, at which time pollen tubes have penetrated much of the length of the style but have not yet entered the ovary. At least 11 major proteins from pollinated ovaries had no detectable counterparts in unpollinated ovaries. These showed a range of molecular mass and pI. For peroxidase isozyme assays, ovaries were sampled at 0, 12, 24, 36 and 48 h after pollination. At 45–50 h, pollen tubes were beginning to enter the top of the ovary but could still be separated from the ovules and placenta during sampling. Ovules and placentae from unpollinated pistils showed only one form of peroxidase, whereas those from pollinated pistils showed additional isozymes at pH 5.4 and pH 10.0. Both new isozymes increased in staining intensity over the first 36 h after pollination. At 48 h, however, the acidic peroxidase had continued to increase, while the basic component had declined so as to be barely detectable. The observations are discussed in relation to accumulating evidence that some form of pollination-induced signal reaches the ovary ahead of the advancing pollen tubes. The nature of this signal and possible involvement of peroxidases are also briefly discussed.

Purification of a 24 kD parasitism‐specific hemolymph protein from pharate pupae of the caribbean fruit fly, Anastrepha suspensa

We report purification of a 24 kD parasitism-specific protein (24 kD PSP) from pharate pupal hemolymph of the Caribbean fruit fly, Anastrepha suspensa, after parasitization by the braconid wasp, Diachasmimorpha (= Biosteres) longicaudata. We previously utilized isoelectric focusing (IEF) and two-dimensional (2-D) gel electrophoresis to demonstrate that the 24 kD PSP consists of two variants with pl 6.7 (more abundant) and pl 6.3. Purification of the more abundant 24 kD PSP variant was accomplished by Concanavalin A (Con A) sepharose B affinity chromatography followed by DEAE column chromatography. A second protocol, utilizing wheat germ agglutinin (WGA) sepharose 6MB affinity chromatography between the ConA and DEAE chromatographic steps, resulted in the purification of a partially deglycosylated form of the 24 kD PSP which retained its immunoreactivity with anti-PSP serum but which exhibited a greater relative migration in sodium dodecyl sulfate (SDS)-PAGE than the pl 6.7 24 kD PSP variant. For structural studies both 24 kD PSP variants were purified from whole hemolymph by flat bed IEF followed by SDS-PAGE. Peptide cleavage profiles in 1-D SDS-PAGE after treatment with BNPS-skatole, CNBr, and endproteinases Lys-C and Asp-N were identical for both 24 kD PSP variants. Primary N-terminus sequences of at least the first 20 amino acid residues of both variants were identical. A secondary sequence of five amino acids residues was detected in both variants at Thr, the seventh amino acid residue from the N-terminus of the primary sequence. These data indicate that both 24 kD PSPs are glycoforms of a branched, apparently homogeneous polypeptide.

Co-purification of a heat-stable antioxidant with the superoxide dismutase activity from dried peas

A heat-stable antioxidant component was observed to co-purify with the superoxide dismutase (SOD) activity from dried peas. The heat-stable component co-purified by size (gel filtration on Sephadex G-75) and by charge (flat-bed isoelectric focusing (IEF) and Rotofor IEF) coincidently with the SOD activity. Throughout the purification procedure the SOD fractions all exhibited heat-stable antioxidant activity. The autoxidation of linoleic acid in the presence of the pea SOD fraction was delayed for more than four weeks. Purified pea SOD may provide a novel protein-bound, thermostable natural oxidant.

The amino-acid sequence of two isoforms of alpha-lactalbumin from donkey (Equus asinus) milk is identical.

The complete primary structure of donkey alpha-lactalbumin A and B has been determined by sequencing of the peptides obtained after tryptic, Glu-C proteinase or cyanogen bromide cleavage. Although preparative purification of alpha-lactalbumin by flat-bed isoelectric focusing gave two protein fractions A and B, their amino-acid sequence revealed no differences. Donkey alpha-lactalbumin shows two, four and five differences in comparison to the horse alpha-lactalbumin A, B and C. Thus donkey alpha-lactalbumin is homogeneous and belongs to the horse A-type variant.

Isoelectric Heterogeneity of Human Prorenin (Inactive Renin) in Body Fluids

Using flat bed isoelectric focusing, inactive renins from various body fluids were compared. Normal plasma inactive renin demonstrated six consistent peaks at pH 4.98, 5.14, 5.29, 5.47, 5.63 and 5.91, the largest being 5.29, 5.47 and 5.63. The isoelectric pattern of ovarian follicular fluid inactive renin was similar, with slight shifts at major peaks to a higher pH. Pregnancy plasma had inactive renin patterns like normal human plasma as did pleural fluid, lymphocoele fluid and plasma from anephric humans. Amniotic fluid inactive renin showed a markedly different pattern with major peaks at pH 5.00, 5.16 and 5.30. Fetal plasma (cord blood) also showed differences with only four peaks at 5.24, 5.40, 5.58 and 5.83. We conclude that human prorenin isoelectric patterns are similar for plasma, ovarian and most tissue fluid prorenins, but different for fetal plasma and amniotic fluid suggesting that these forms of renin do not cross the placental barrier.

The chromosomal location of genes encoding NADH dehydrogenase isozymes in hexaploid wheat and related species

Analysis of NADH dehydrogenase isozymes in hexaploid wheat (Triticum aestivum) by flat-bed isoelectric focusing revealed a genetic system involving four sets of Ndh loci. The Ndh-A1 locus, previously identified on chromosome arm 4AL, was confirmed. Homoeoloci Ndh-R1 in rye, Ndh-H1 in barley, Ndh-Hch1 in Hordeum chilense, Ndh-Sl1 in Aegilops sharonensis, and Ndh-V1 in Dasypyrum villosum were also identified. The Ndh-2 loci, encoding monomeric isozymes, were found in the short arms of homoeologous group 7 chromosomes. Homoeoloci Ndh-A2, Ndh-D2, and Ndh-R2 are described. The Ndh-3 set, encoding multimeric isozymes, were found on each of the homoeologous group 3 chromosome long arms. An Ndh-B3b variant and homoeoloci Ndh-R3 (on chromosome arm 6RL), Ndh-H3, and Ndh-Sl3 are described. The Ndh-4 set, encoding monomeric isozymes for which no varietal variation was found, were identified on the short arms of group 3 chromosomes. Ndh-A4, Ndh-B4, Ndh-H4, Ndh-R4, and Ndh-E4 (Agropyron elongatum) homoeoloci are described.Key words: NADH dehydrogenase, isozymes, wheat, Triticeae.

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c′, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c′ was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.

THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING.

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.

Evidence for the lack of exo-cellobiohydrolase activity in the cellulase system of Trichoderma reesei QM 9414

A cellulolytic enzyme (pI 4.3) was isolated from culture filtrates of Trichoderma reesei QM 9414 by column chromatography and preparative flat bed isoelectric focusing. The focusing experiments were carried out using an extremely shallow pH gradient (pH 3.7–4.6), which made it possible to recover one single protein band out of numerous Trichoderma isoenzymes and/or multi-enzyme complexes in the acidic pH region. The purified hydrolase is homogeneous as assessed by analytical isoelectric focusing, SDS gel electrophoresis, and titration curve analysis under detergent/ urea conditions. It was found to have an apparent molar mass of 68 000 g mol −1 (SDS-PAGE) and showed its optimum activity towards cellotetraose at pH 4 and 60°C. It is a glycoprotein with a carbohydrate content of 27% (as glucose). The hydrolase does not act with an endo-mechanism (CMC as substrate) and splits off mainly cellobiose from microcrystalline cellulose. In the current literature the enzyme would thus be termed an exo-cellobiohydrolase [1,4-β-D-glucan cellobiohydrolase (CBH), EC 3.2.1.91]. However, when the enzyme's action pattern was further evaluated using defined soluble cellodextrins (DP 2–7) as substrates, it became obvious from the recorded concentration-time course data that the enzyme does not exclusively split off cellobiose units from the oligomers. In conclusion, we do not believe that a cellobiohydrolase analogous to β-amylase (EC 3.2.1.2) exists, at least in the cellulase system of Trichoderma reesei. The term CBH or cellobiohydrolase should thus not be used any longer in the literature.

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva.

Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15,000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10-20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the alpha- and beta-charge isomers of pheromaxein which were shown to have isoelectric points of 4.78 and 5.35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5 alpha- and 5 beta-androstane series also showed some binding to pheromaxein, i.e. 17 beta-hydroxy-5 alpha-androstan-3-one (19.2%), with 5 alpha-androstan-3-one, which has a similar urinous odour to 5 alpha-androst-16-en-3-one, showing the greatest binding (42.6%) relative to 5 alpha-androst-16-en-3-one (100%).

Purification to homogeneity and biochemical characterization of two suppressor factors from human malignant T-cells

A cell clone (GI-CO-T-9) derived from a long term T-cell culture (PF-382), established from a patient affected by acute T-lymphoblastic leukemia (T-ALL), was selected for the presence in the culture medium of factors suppressing T-cell proliferation. The crude supernatant has been subjected to a multi-step chromatographic fractioning, including: preparative gel permeation, anion exchange, and hydrophobic interaction High Performance Liquid Chromatography (HPLC). The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), revealing single bands of 115 Kd and 80 Kd. The isoelectric points (pI), determined by flat-bed isoelectricfocusing, were 7.4 for High Molecular Weight Suppressor Factor (HMWSF) and 3.5–3.6 for Low Molecular Weight Suppressor Factor (LMWSF).

Functional and biochemical characterization of B-cell differentiation factor (BCDF) produced by an HTLV-I-transformed human T-cell clone and demonstration of specific binding of the factor to a BCDF responsive cell line

We have previously described YA2, a human T-cell clone that secretes B-cell differentiation factor (BCDF) but not B-cell growth factor (BCGF). The BCDFs secreted by YA2 and HTLV-I-transformed YA2 (TYA2) were functionally similar in their ability to stimulate Ig secretion by Staphylococcus aureus Cowan strain I-activated B cells and IgM secretion by SKW6.4 cells. In addition, they were biochemically similar with a MW of 30 kDa by high-performance liquid chromatography (HPLC) sieving, and a p I of 6.0–6.8 by isoelectric focusing. The BCDF activity was not blocked by antibodies to interleukin 2 and BCGF. BCDF was purified from TYA2 supernatant by sequential media protein immunoadsorption, flat bed isoelectric focusing, HPLC TSK 2000 sieving, and repeated immunoadsorption and was then iodinated. The iodinated material had functional BCDF activity and migrated to a single band at MW 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at p I of 6.8 by polyacrylamide gel isoelectric focusing. 125I-BCDF purified in this manner bound specifically to a BCDF-responsive cell line and not to phytohemagglutinin-activated T cells. 125I binding to the BCDF-responsive cell line was competitively inhibitable by the addition of cold BCDF. Thus we have purified and characterized a factor with BCDF activity and demonstrated that this factor binds specifically to a BCDF-responsive cell line.

α-Amylase and β-amylase homoeoloci in species related to wheat

SummaryA study of α-amylase isozyme patterns from gibberellin-induced endosperms of wheat-alien genotypes (amphiploid, addition and substitution lines) resolved by flat-bed isoelectric focusing identified homoeoloci for α-Amy-1(malt α-AMY-1 genes) on chromosomes 6H ofHordeum vulgare, 6RL ofSecale cereale, 6RmofS. montanumand 6E ofAgropyron elongatum. Homoeoloci for α-Amy-2(green α-AMY-2 genes) were identified on chromosomes 7HchL ofHordeum chilense, 7RL ofSecale cereale, 7SbofAegilops bicornis, 7U ofAe. umbellulataand 7EL ofAgropyron elongatum. Analysis of mature grain β-amylase identified β-Amy-1loci on chromosomes 4H ofH.vulgare, 4HchofH. chilense, 4S1ofAe. sharonensisandAe. longissimaand β-Amy-2loci on chromosomes 5RL ofS. cerealeand 5U ofAe. umbellulata. These gene locations provide further evidence for the homoeology of the alien chromosomes with wheat and for the conservation of gene synteny among wheat and its relatives.