Altered gene expression of c-myc, epidermal growth factor receptor, transforming growth factor-alpha, and c-erb-B2 in an immortalized human breast epithelial cell line, HMT-3522, is associated with decreased growth factor requirements.

Activation of protooncogenes and constitutive secretion of autocrine growth factors are thought to be involved in the uncontrolled growth of cancer cells. We have attempted to elucidate the role of oncogenes and growth factors in the premalignant progression of human breast epithelial cells by using an immortalized, nontumorigenic, near-diploid human mammary epithelial cell line, HMT-3522, derived from a fibrocystic lesion and established in our laboratory. During propagation in tissue culture, the growth factor requirements of the HMT-3522 cells decreased simultaneously with an amplification and overexpression of the c-myc protooncogene. Other protooncogenes related to human breast cancer were unaltered with regard to gene copy number and expression. In passage 118, in which the most important growth factor still was epidermal growth factor (EGF), we were able to isolate an EGF-independent subline (S2). The EGF independence of S2 was accompanied by an overexpression of the mRNAs for epidermal growth factor receptor (EGF-R), transforming growth factor-alpha, and c-erb-B2 as compared to the EGF-dependent subline (S1). Moreover, by application of a blocking anti-EGF-R antibody, growth of S2 cells in EGF-free medium was inhibited significantly, indicating that EGF-R was involved in an autocrine loop probably with transforming growth factor-alpha as ligand. Neither the late passages of S1 cells nor S2 cells were tumorigenic after subcutaneous transplantation to athymic mice. Our results indicate that c-myc amplification and overexpression are correlated with a decreased requirement for growth factors. Even when these alterations are combined with immortalization and EGF independence, they are insufficient for malignant transformation of these human breast epithelial cells.

Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909S1 and HMT-3909S8, established in serum-free medium.

In a serum-free medium we have established two new human breast carcinoma cell lines from a single primary tumor. Cultures were maintained on chemically defined medium CDM3 or on minor modifications of this medium, Dulbecco's modified Eagle medium-Ham's F12 supplemented with epidermal growth factor, insulin, transferrin, estradiol, hydrocortisone, triiodothyronine, cyclic AMP, phosphoethanolamine, ethanolamine, fibronectin, fetuin, ascorbic acid, bovine serum albumin, and trace element salts including selenite (Petersen and van Deurs, Cancer Res., 47: 856-866, 1987). Primary cultures comprised both NADPH-neotetrazolium reductase-positive carcinoma cells and NADPH-neotetrazolium reductase-negative cells of stromal appearance, as well as normal epithelial cells (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986). In subsequent passages the cells were monitored exclusively using the tumorigenicity assay on nude mice. Two cell lines, one nontumorigenic, HMT-3909S1, and one tumorigenic, HMT-3909S8, were selected from the primary cultures. Selection of S8 through subline S4 required transient supplementation of CDM3 with fetal calf serum. Permanent lines S1 and S8 were maintained on serum-free medium. Further characterization of the two cell lines in terms of normal breast gland differentiation (Petersen and van Deurs, Differentiation, 39: 197-215, 1988) was carried out using immunocytochemistry, immunochemistry, electron microscopy, and cytogenetics. S8 appeared to be identical with the NADPH-neotetrazolium reductase-positive carcinoma cells of the primary cultures, with a particular subpopulation of carcinoma cells in the tumor of origin, and with the tumorigenic cells of the nude mice. This subline was aneuploid, typically epithelial in morphology, and expressed keratins K8 and K18 and the glycoprotein MAM-6, typical of luminal epithelial cells in the normal breast gland. Subline S1 appeared more like the elongated cells in the primary cultures and like a second subpopulation of cells in the carcinoma of origin. However, S1 cells were in fact epithelial, since they expressed keratins. Also, S1 cells seemed to be a triploidation of a cell with close resemblance to S4, while only few cytogenetic differences were found between S4 and S8, suggesting an origin of S1 and S8 via S4 from a single hypothetical stem cell.

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.

Effect of insulin on growth and expression of smooth muscle isoactin in human breast gland myoepithelial cells in a chemically defined culture system.

Myoepithelial cells express both epithelial and stromal (smooth muscle) cell characters. Moreover, while separating the luminal (secretory) epithelial cells from the connective tissue in normal breast glands, myoepithelial cells apparently disappear in invasive carcinomas, or their phenotypic characteristics become down-regulated. In the present study we have used a chemically defined culture model system to study how expression of smooth muscle isoforms of actin in myoepithelial cells is influenced by insulin by using immunoblotting, immunofluorescence and electron microscopy. We show that in the absence of insulin, myoepithelial cells do not proliferate but exhibit a differentiated phenotype. Hence, they contain distinct bundles of actin filaments and also numerous caveolae at the cell surface. In contrast, with insulin in the medium, cell proliferation increases dramatically. Concomitantly the smooth muscle actin expression and the associated caveolae disappear within a week. However, other cytoskeletal proteins such as keratins and vimentin are expressed no matter whether insulin is absent or present.

A simple immunoblotting method after separation of proteins in agarose gel.

A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence of multiple molecular forms of the complement factors C3 and factor B in serum from 2 species, man and chicken, after electrophoretic separation in agarose. We have also demonstrated the usefulness of the method for determining the isoelectric point of proteins after isoelectric focusing in agarose.

Regulation of the secretion of proteins synthesized by the human breast cancer cell line, MCF-7.

In two sublines of the estrogen receptor-positive human breast cancer cell line, MCF-7, propagated with 0.5% fetal calf serum (FCS) (subline 0.5) and without serum (subline 9), respectively, addition of 10% newborn calf serum (NCS) inhibits cell proliferation and stimulates the secretion of a 42 kDa protein. Both with and without NCS, estradiol (E2) stimulates the secretion of a 61 kDa and a 52 kDa protein in subline 9, and an additional third protein (66 kDa) is stimulated in subline 0.5. E2 inhibits the synthesis of the 42 kDa protein in both sublines. Different cell proliferation rates of MCF-7 cells can be established in cultures with 10% NCS and varying concentrations of E2. In such experiments final cell number after 6 days is positively correlated to the relative amount of the 66 kDa, the 61 kDa and the 52 kDa proteins, whereas the relative amount of the 42 kDa protein is negatively correlated to the final cell number. These results indicate that the 52 kDa, the 61 kDa and the 66 kDa proteins may act as positive growth factors, whereas the 42 kDa protein may act as a negative growth factor. The 42 kDa, the 52 kDa and the 61 kDa proteins are apparently glycoproteins, and the 66 kDa protein is not glycosylated. The 61 kDa protein is immunoreactive with antitrypsin antibodies.

EPR and DNP properties of certain novel single electron contrast agents intended for oximetric imaging.

Parameters of relevance to oximetry with Overhauser magnetic resonance imaging (OMRI) have been measured for three single electron contrast agents of the triphenylmethyl type. The single electron contrast agents are stable and water soluble. Magnetic resonance properties of the agents have been examined with electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and dynamic nuclear polarization (DNP) at 9.5 mT in water, isotonic saline, plasma, and blood at 23 and 37 degreesC. The relaxivities of the agents are about 0.2-0.4 mM-1s-1 and the DNP enhancements extrapolate close to the dipolar limit. The agents have a single, narrow EPR line, which is analyzed as a Voigt function. The linewidth is measured as a function of the agent concentration and the oxygen concentration. The concentration broadenings are about 1-3 microT/mM and the Lorentzian linewidths at infinite dilution are less than 1 microT in water at room temperature. The longitudinal electron spin relaxation rate is calculated from the DNP enhancement curves. The oxygen broadening in water is about 50 microT/mM O2 at 37 degreesC. These agents have good properties for oximetry with OMRI.

Serum albumin as a modulator on growth of the human breast cancer cell line, MCF-7.

The growth of the estrogen responsive human breast cancer cell line, MCF-7, is inhibited by high serum concentrations, and this growth inhibition can be abolished by estradiol (E2). To investigate this inhibitory phenomenon further, we decided to purify the inhibitory factor from newborn calf serum (NCS). After the use of various fractionation methods, we found that inhibitory activity in NCS was exclusively expressed by albumin containing fractions. The inhibitory potential of several commercial bovine serum albumin (BSA) preparations and one human serum albumin preparation were analysed. They all exerted inhibitory activity comparable to that of NCS, and BSA inhibited MCF-7 cell proliferation in a concentration-dependent manner similar to that of NCS. Albumin itself or a contaminating factor in the albumin preparations seemed to be responsible for the growth inhibition. It could be excluded that the growth inhibitor TGF-beta, known to be present in serum, was the factor which inhibited MCF-7 cell proliferation. We separated contaminating proteins from albumin by gel filtration of a BSA preparation, revealing that neither low mol.wt nor high mol.wt proteins in the preparation exerted any significant growth inhibitory activity. NCS and BSA affected the secretion of specific proteins from MCF-7 cells similarly, when grown with or without E2. In conclusion, we assume that albumin is the factor in serum exerting a growth inhibition which can be reversed by E2. Our results indicate that albumin may affect cell proliferation by modulating the activities of autocrine growth regulatory factors.

Mannan-binding lectin and healing of a radiation-induced chronic ulcer--a case report on mannan-binding lectin replacement therapy.

Mannan-binding lectin is an important component of innate immunity, and insufficiency is associated with several clinical disorders. Recently, experimental replacement therapy with plasma-derived mannan-binding lectin has become an option. The current article presents the case of a patient with an insufficient level of mannan-binding lectin and a chronic radiation-induced ulcer following the treatment of breast cancer. After 15 months of initially conservative treatment and thereafter plastic surgery, the healing was still impaired with necrosis in the periphery of the ulcer. Immunological work-up of the patient revealed pronounced insufficiency of mannan-binding lectin. Following a 6-week experimental intravenous treatment with mannan-binding lectin purified from human plasma, that is, 0.2-0.3 mg mannan-binding lectin per kg body weight twice a week, the defect was completely healed. We suggest that deficiency of mannan-binding lectin can explain cases of otherwise unexplained impaired healing, and that replacement therapy is considered in such cases.

Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics.

BACKGROUND AND OBJECTIVES: Mannan-binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro-organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma-derived MBL is a promising treatment of diseases associated with MBL deficiency. A first-generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second-generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product. MATERIALS AND METHODS: Carbohydrate-based gels were comparatively screened as affinity matrices. MBL was extracted from fraction III, and affinity purified on a Superdex 200 pg column. The eluted material underwent two virus reduction steps: filtration through Planova 20N and solvent/detergent treatment. It was further purified by anion-exchange and gel-filtration chromatography. The affinity eluate and the final MBL fraction were characterized by protein chemical, immunological, and functional assays. RESULTS: In production scale, Superdex 200 pg was found to be superior to other carbohydrate-based matrices, and MBL was affinity purified from fraction III with a yield of 70%. The viral safety was increased by performing a nanofiltration of the affinity eluate through Planova 20N with a minimal loss of MBL. The purity of the final MBL fraction was 53% excluding the MBL-associated serine proteases (MASP). The product consisted of high-oligomeric MBL, with two dominating forms, and with MASP-1, -2, -3 and 19 kDa MBL-associated protein (MAp19). Only a few protein impurities were present, the major being alpha2-macroglobulin. MBL formed complexes with alpha2-macroglobulin bridged by MASP-1 covalently attached to the latter. The functional activity, assessed by mannan-binding activity and opsonic function, was intact, whereas half of the C4 activating capacity was lost during the production process. CONCLUSION: A second-generation MBL process was developed with an average yield of 50%. It was possible to nanofilter the MBL-MASP complexes through Planova 20N with only a minor loss resulting in an increased safety profile of this MBL product.

Comparison of rocket and crossed immuno-electrophoresis assays for determination of the level of actin complexing of Gc globulin.

OBJECTIVE: Gc globulin (vitamin D-binding protein) is a component of the extracellular actin scavenger system. The level of Gc globulin is reduced in patients with fulminant hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. Owing to a large increase in the turnover of Gc globulin upon complex formation with actin, it may be important to determine both the total Gc globulin concentration and the degree of complexing with actin for estimating the clinical prognosis of a patient. For this reason, we have compared a crossed immuno-electrophoresis method (CIE), suitable for visualizing the degree of complexing with actin, with a rocket immuno-electrophoresis method (RIE), previously used for determination of the complex degree. MATERIAL AND METHODS: Sera from healthy donors and from patients with acetaminophen-induced liver disease or trauma were investigated using CIE, RIE and enzyme-linked immunosorbent assay (ELISA). RESULTS: Using the CIE, no Gc globulin-actin complexes were detected among healthy donors. Complexes were present in 21 of 39 patients with liver disease and 3 of 37 trauma patients. High complex ratios (> 20 %) were found in 6 of 7 patients with hepatic encephalopathy. Using the RIE, complexes were detected in most samples. CONCLUSION: The results show that the CIE method may be used for determining the degree of actin complexing in conjunction with ELISA or RIE in determining the levels of total Gc globulin.

Mannan-binding lectin (MBL) production from human plasma.

Individuals with low levels of mannan-binding lectin (MBL) appear to be susceptible to infectious diseases. This suggests that substitution therapy with MBL might be a beneficial treatment of patients with MBL deficiency. A production process for an MBL product has been developed from a fraction II+III precipitate obtained by ethanol fractionation of plasma. The MBL process includes three chromatographic steps, where the first and key step is affinity chromatography on a cross-linked agarose matrix selecting for oligomeric, carbohydrate-binding MBL. The yield from the production process is about 25% of the plasma MBL content, and the purity is about 65%. The MBL product shows mannan-binding activity and complement-activating ability. A safety study has shown this plasma-derived MBL to be safe and well tolerated in adult MBL-deficient volunteers.

Purification of chicken C3 and a structural and functional characterization.

A major plasma protein from chicken, analogous to mammalian complement component C3, was purified by the removal of plasminogen, precipitation with polyethyleneglycol, and ion-exchange chromatography. Purification was guided by a rabbit antiserum specific to chicken C3. The yield of native C3 was 27%, and purity and functional activity was assessed by SDS-PAGE, immunoprecipitation techniques, and the ability of the purified C3 to restore the haemolytic activity of C3-depleted chicken serum. Monoclonal antibodies were raised against purified chicken C3. These antibodies were characterized and used to prepare an immunosorbent column to deplete chicken plasma specifically of C3. Chicken C3 has a mol.wt of 185,000-195,000 and a two-chain structure with an alpha chain (118,000) and beta chain (68,000). Complement activation leads to changes in the electrophoretic mobility of chicken C3 and to a decrease in mol.wt to 144,000 corresponding to the release of a 15,000 C3a and a 34,000 C3d/C3dg fragment. Chicken C3 exists in multiple molecular forms with pI values of 6.4-6.6. A genetic polymorphism of chicken C3 based on electrophoretic mobility has not yet been detected after analysis of more than 500 individuals. The function of chicken C3 is dependent on a reactive thioester because treatment of purified chicken C3 with methylamine causes functional inactivation of C3.

Reactive arthritis and serum levels of mannose binding lectin -- lack of association.

The purpose was to evaluate the possible association of serum mannose binding lectin (s-MBL) levels on type of triggering microbe, duration of diarrhoea, incidence and course of reactive arthritis (ReA) caused by Salmonella, Yersinia and Campylobacter. Sixty patients with ReA of 1-228 months duration, 173 patients with ReA or uncomplicated enterocolitis caused by Campylobacter, 226 sera from patients with elevated antibody levels against Salmonella, Yersinia or Campylobacter, and 114 blood donors were tested for s-MBL using ELISA technique, both direct mannan binding assay and sandwich ELISA. s-MBL was compared with C-reactive protein (CRP) levels and with the ability of activating complement C4. Among the 114 donors 9% had s-MBL <50 microg/l, 16% had from 50-500 microg/l and 75% had >500 microg/l. The distribution of s-MBL levels in the three-patient groups did not differ significantly from the controls. There were no indications that low s-MBL was associated with prolonged duration of arthritis, diarrhoea or individual bacterial infections. The two MBL assays were comparable with respect to serum concentrations, indicating that the actual circulating MBL was also functionally active. s-MBL exhibited acute phase reactant behaviour and correlated to CRP level, but only in patients with s-MBL concentrations exceeding 1000 microg/l. MBL in 10 randomly selected ReA sera were tested for the ability to activate complement C4. The results did not differ from those of donor controls. This study demonstrates that the distributions of s-MBL levels in serum among patients with ReA are not different from donor controls. The course, outcome or triggering bacteria are not associated with a particular level of s-MBL.

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.

Elevated serum levels of fetal antigen 1,a member of the epidermal growth factor superfamily, in patients with small cell lung cancer.

Serum levels of fetal antigen 1 (FA1) were quantified pretherapeutically in 16 patients with pneumonia, 30 patients with small cell lung cancer (SCLC) and 10 patients with non-small cell lung cancer (NSCLC) and compared to the normal reference interval (n = 177). Serum FA1 levels were significantly elevated in SCLC (p < 0. 0001) but not in pneumonia or NSCLC (p = 0.1467 and p = 0.3262, respectively). With the 95th centile of the normal range as cutoff level the sensitivity for SCLC was 43% and the specificity 96%. There was no correlation to neuron-specific enolase levels or to the diagnosis of limited/extensive disease. Immunohistochemical analysis of a biopsy from 1 SCLC patient with an elevated serum FA1 also showed the presence of FA1 in tumor cells. FA1 in serum from SCLC patients was identical to that of FA1 in normal serum/amniotic fluid with respect to size distribution and also revealed a reaction of immunological identity with FA1 in amniotic fluid.

The relationship between physical and genetic distances at the Hor1 and Hor2 loci of barley estimated by two-colour fluorescent in situ hybridization.

The hordeins are the major class of storage proteins in barley. They are encoded by multigene families. The B- and C-hordein loci have been mapped physically to the distal end of chromosome 5 (1I) of cultivated barley by fluorescent in situ hybridization. Based on measurements of chromosomal distances between the two hordein loci, the relationship between genetic and physical distances has been estimated to be about 1 mega base pairs per centiMorgan. This is four times higher than the mean value for the barley genome as a whole and confirms the tendency to increased recombination in distal chromosome regions. The resolving power of two-colour FISH is discussed. It is concluded that the method is suitable for estimating the relationship between genetic and physical distances of regions of about 10 Mbp or larger.

Elimination and duplication of particular Hordeum vulgare chromosomes in aneuploid interspecific Hordeum hybrids.

Seeds formed in crosses Hordeum lechleri (6x) x H. vulgare (2x and 4x), H. arizonicum (6x) x H. v. (2x), H. parodii (6x) x H. v. (2x), and H. tetraploidum (4x) x H. v. (2x) produced plants at high or rather high frequencies through embryo rescue. Giemsa C-banding patterns were used to analyze chromosomal constitutions and chromosomal locations on the methaphase plate. Among 100 plants obtained from H. vulgare (2x) crosses, 32 plants were aneuploid with 2n=29 (1), 28 (3), 27 (13), 26 (5), 25 (4), 24 (4), or 22 (2); 50 were euploid (12 analyzed), and 18 were polyhaploid (5 analyzed). Four plants had two sectors differing in chromosome number. Two of four hybrids with H. vulgare (4x) were euploid and two were aneuploid. Parental genomes were concentrically arranged with that of H. vulgare always found closest to the metaphase centre. Many plants showed a certain level of intraplant variation in chromosome numbers. Except for one H. vulgare (4x) hybrids, this variation was restricted to peripherally located non-H. vulgare genomes. This may reflect a less firm attachment of the chromosomes from these genomes to the spindle. Interplant variation in chromosome numbers was due to the permanent elimination or, far less common, duplication of the centrally located H. vulgare chromosomes in all 34 aneuploids, and in a few also to loss/gain of non-H, vulgare chromosomes. This selective elimination of chromosomes of the centrally located genome contrasts conditions found in diploid interspecific hybrids, which eliminate the peripherally located genome. The difference is attributed to changed "genomic ratios'. Derivatives of various H. vulgare lines were differently distributed among euploid hybrids, aneuploids, and polyhaploids. Chromosomal constitutions of hypoploid hybrids revealed a preferential elimination of H. vulgare chromosomes 1, 5, 6, and 7, but did not support the idea that H. vulgare chromosomes should be lost in a specific order. H. vulgare SAT-chromosomes 6 and 7 showed nucleolar dominance. Aneuploidy is ascribed to the same chromosome elimination mechanism that produces haploids in cross-combinations with H. vulgare (2x). The findings have implications for the utilization of interspecific Hordeum hybrids.

Karyotypes of Elymus scabrifolius (Poaceae: Triticeae) from South America studied by banding techniques and in situ hybridization.

Karyotypes of 4 accessions of Elymus scabrifolius (2n = 4x = 28) were investigated by Giemsa C- and N-banding, GAA-banding (one accession), AgNO3-staining and in situ hybridization with the rDNA probe pTa71. Two additional accessions were studied in less detail. The chromosomes were large (9-14 microns). The complements included 11 pairs of metacentrics, one with conspicuous satellites on the short arms, and 3 pairs of submetacentrics. Two of 4 accessions from Eastern Argentina and Uruguay had minute or small satellites on a submetacentric pair. No such satellites were observed in the other two accessions. In two accessions from the Cordoba province, a non-homologous submetacentric pair had very long satellites. AgNO3-staining established the presence of 4 nucleoli, two larger and two small ones, in 5 accessions. The C-banding patterns comprised from one to 12 conspicuous bands per chromosome at no preferential positions. The amount of constitutive heterochromatin (19-21%) was the highest hitherto established in the Triticeae. Similarities in banding patterns and chromosome morphology identified homologous and discriminated between non-homologous chromosomes within and, except for two chromosomes, between plants. Heteromorphic chromosome pairs were identified in satellite-carrying chromosomes only. N-banding produced conspicuous bands overall at the same positions as C-banding. GAA-banding patterns were similar to N-banding patterns. The rDNA probe hybridized to chromosome segments at nucleolar constrictions only. The production of C- and N-banding patterns in both genomes of E. scabrifolius suggests the presence of two H genomes and the absence of the pivotal St genome of Elymus. On account of the uncertain identity of one genome, and the overall similar gross morphology of E. scabrifolius and other tetraploid South American species referred to Elymus, E. scabrifolius is retained in Elymus.

Chromosomal locations of four minor rDNA loci and a marker microsatellite sequence in barley.

Four minor rDNA loci have been mapped physically to barley (Hordeum vulgare L.) chromosomes 1 (7l), 2 (2l), 4 (4l), and 5 (1l) by a two-step in situ hybridization procedure including a GAA microsatellite sequence. Reprobing with the microsatellite resulted in a distinct banding pattern, resembling the C-banding pattern, which enabled unequivocal chromosome identification. This study suggests that gene mapping accuracy may be improved by using probes with well-characterized and narrow hybridization sites as cytological markers which are situated close to the gene locus. One of the rDNA loci is located about 54% out on the short arm of chromosome 4 and it has not previously been reported in barley. We have designated the new locus Nor-l6. rDNA loci on homoeologous group 4 chromosomes have not yet been reported in other Triticeae species. The origin of these 4 minor rDNA loci is discussed in relation to their equilocal distribution on the chromosomes.