Immunocytochemical localization of two myosins within the same muslce cells in Caenorhabditis elegans.

Nematodes synthesize two major classes of myosin heavy chains. These heavy chains associate to form only homodimeric myosin molecules, and these myosin homodimers are anti-genically different from one another (Schachat, Garcea and Epstein, 1978). The two myosins may be designated unc-54 myosin, since this species is altered in mutants of the unc-54 locus, and non-unc-54 myosin, since this class is not affected in unc-54 mutants. We present here experiments in which specific anti-myosin IgG and anti-unc-54 myosin IgG are used to locate the two myosins within the same body-wall muscle cells of Caenorhabditis elegans. These results are necessary for further evaluation of the possible functions of the two myosin homodimers in the thick filaments of these muscles.Myosin can be localized to all body-wall and pharyngeal muscle cells using anti-myosin antibody. In longitudinal sections of body-wall muscle, the staining with anti-myosin coincides with the birefringence of A bands that contain thick filaments. Anti-unc-54 myosin stains all body-wall A bands uniformly but does not react with the pharynx. This result demonstrates that unc-54 is located exclusively in body-wall muscle cells of the wild-type strain N2. Non-unc-54 myosin is localized with anti-myosin in all body-wall muscle cells of the unc-54 null mutant E190, as expected; however, unc-54 myosin could not be detected by anti-unc-54 myosin antibody in this mutant.Since we can localize unc-54 myosin and non-unc-54 myosin in all body-wall muscle cells of wild-type and E190, respectively, we conclude that the two myosins must be present in the same muscle cells. In addition, since unc-54 myosin is located in all body-wall A bands, at least some sarcomeres must contain both myosins. This conclusion is consistent with the observations of Garcea, Schachat and Epstein (1978) that wild-type and E190 synthesize similar amounts of non-unc-54 myosin. Within the limits of resolution of our methods, unc-54 myosin is distributed throughout body-wall A bands. We conclude, therefore, that the majority of thick filaments within these A bands must contain unc-54 myosin along their entire length. Possible roles for unc-54 and non-unc-54 myosins in the assembly and organization of thick filaments are discussed.     

Myosins exist as homodimers of heavy chains: demonstration with specific antibody purified by nematode mutant myosin affinity chromatography.

The body-walls of Caenorhabditis elegans contain two different myosin heavy chains (Epstein, Waterston and Brenner, 1974) that associate to form at least two species of myosin (Schachat, Harris and Epstein, 1977a). To better define the distribution of these heavy chains in myosin molecules, we have characterized the myosin of C. elegans by immunochemical methods. Specific, precipitating anti-myosin antibody has been prepared in rabbits using highly purified nematode myosin as the immunogen. The difference in reactivity of the anti-myosin antibody with wild-type myosin containing both kinds of heavy chains (designated unc-54 and non-unc-54 heavy chains on the basis of genetic specification) and myosin from the mutant E190 that lacks unc-54 heavy chains Indicates that there are antigenic differences between myosin molecules containing unc-54 heavy chains and myosin molecules containing only non-unc-54 heavy chains. Antibody specific for the unc-54 myosin determinants has been prepared by the immunoadsorption of anti-myosin antibody with E190 myosin. This specific anti-unc-54 myosin antibody precipitates myosin that contains only unc-54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, we could detect no heterodimeric myosin molecules containing both unc-54 and non-unc-54 heavy chains. The body-wall myosins of C. elegans therefore exist only as homodimers of either class of heavy chain.This specific anti-unc-54 myosin antibody promises to be a valuable tool in elucidating the role of two myosins in body-wall muscle and in molecular characterizations of mutant myosins in C. elegans. We report here the use of this antibody to detect antigenic differences between unc-54 myosin from the wild-type and the muscle mutant E675. In conjunction with the original anti-myosin antibody, other studies show that both unc-54 and non-unc-54 myosins exist within the same body-wall muscle cells (Mackenzie, Schachat and Epstein, 1978) and that both myosins are coordinately synthesized during muscle development in C. elegans (Garcea, Schachat and Epstein, 1978). We discuss the implications of the self-association of unc-54 and non-unc-54 myosin heavy chains into homodimeric myosins within the same body-wall muscles with respect to the assembly of thick filaments and their organization into a regular lattice.     

Coordinate synthesis of two myosins in wild-type and mutant nematode muscle during larval development

In this paper we examine the role of two myosins in body-wall muscle cells of the nematode Caenorhabditis elegans. Large populations of nematodes are synchronized, and the synthesis and accumulation of myosin heavy chains and total protein are followed through postmitotic larval development. Growth is exponential with time for both the wild-type N2 and the body-wall muscledefective mutant E675, with a longer doubling time for the mutant. Utilizing the electrophoretic polymorphism of the E675 myosin heavy chains, we show that distinguishable classes of heavy chains accumulate differentially throughout development. Immunochemical measurements confirm a similar result in N2. Total myosin heavy chain accumulation is also quantitatively similar for the two strains. Myosin heavy chain relative synthetic rates as determined by pulse-labeling are constant throughout development and are equivalent for the two strains. The final fraction of accumulated unc-54 to total heavy chains of approximately 0.63 equals the constant synthetic fraction of approximately 0.62.Since myosin heavy chain accumulation and relative synthesis are equivalent, we conclude that the turnover of heavy chains is also similar in N2 and E675 despite the extensive structural and functional disruption within body-wall muscle cells of the latter strain. Since the accumulated fraction of unc-54 myosin heavy chains reaches a plateau at the constant synthetic fraction, myosin accumulation In the body-wall muscle cells may be attributed to a constant ratio of synthetic rates of the two body-wall myosin species. The coordinate synthesis of two myosins in the same body-wall muscle cells is discussed.     

Unc45b is essential for early myofibrillogenesis and costamere formation in zebrafish

Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.     

Studies on the myosin molecule from smooth muscle

The myosin molecule was extracted from the smooth muscle parts of horse esophagus and purified by ammonium sulfate fractionation. The schlieren pattern of the sedimentation velocity run showed a very sharp single peak of.5.9. S (s_20,w). Molecular weight of the protein was measured by means of the Archibald and sedimentation equilibrium methods, both in 0.5M KCI buffered by 1/150 M phosphate at pH 7.5 and at 5°C. The values obtained were 6.25 × 10⁵ and 5.81 × 10⁵respectively, for the two methods. The second virial coefficients were 1.1 × 10⁴ and 1.2 × 10^?4 ml/g. Denatured smooth muscle myosin was prepared in a solution of 5M guanidine HC1 containing 0.4 M KC1 and 0.2 M β-mercaptoet hanol buffered at p^H 8.0. The weight-average molecular weight of the denatured smooth muscle myosin was 2.24 × 10⁵ and the second virial coefficient was 7.6 × 10^?4 ml/g. The values described above are in good agreement with those reported for rabbit skeletal myosin with ammonium sulfate fractionation. The molecular dimension of the molecule is estimated as the value for an axial ratio of 100, assuming a rigid rod molecular model for this molecule, both the thermodynamical and hydrodynamical treatment being in a good agreement with this estimation.     

Muscle development in Caenorhabditis elegans: Mutants exhibiting retarded sarcomere construction

We have studied the structural changes within the body-wall muscle cells of Caenorhabditis elegans during postmitotic development. In wild-type, the number of sarcomeres progressively increases, and each sarcomere appears to grow in length and depth continuously during this period. In mature wild-type cells, the anterior-most body-wall muscle cells have 6–7 sarcomeres; the rest have 9–10 sarcomeres per cell. Twelve mutants in the unc-52 II gene exhibit markedly retarded sarcomere construction and progressive paralysis. Several unc-52 mutants, such as the severely paralyzed SU200, produce only 2–3 sarcomeres per body-wall muscle cell, while the other midly paralyzed unc-52 mutants, such as SU250, build 3–4 sarcomeres per muscle cell. Other structures such as the pharynx and even the noncontractile organelles of the body-wall muscle cells do not appear to be structurally or functionally altered. The unc-52 body-wall sarcomeres become moderately disorganized as they are outstripped by cell growth; sufficient order is preserved, however, so that the majority of thick and thin filaments still interdigitate.The myosin heavy chains of SU200 body-wall muscle fail to accumulate normally, while the pharyngeal myosin heavy chains do not appear to be specifically affected. This biochemical result correlates well with the specificity of morphological changes in the mutant. A model is discussed in which the biochemical and morphological deficits are explained by a simple regulatory mechanism.     

Heat-induced Denaturation of Myosin Total Rod

The myosin total rod, which consists of smaller segments of light meromyosin and heavy meromyosin subfragment-2 (HMM S–2), prepared by limited papain digestion of rabbit myosin, was purified by Sepharose-2B column chromatography. The purified total rod was more homogeneous than any previously reported, and the sodium dodecyl sulfate (SDS) gel electrophoretic method yielded a molecular weight of 22?23 × 10⁴ (11?11.5 × 10⁴ × 2).

Transition temperatures of this purified myosin total rod obtained from the melting profile during heating were 47.5 and 55°C. The results of ORD and CD measurements showed almost full reversibility upon cooling after thermal treatment. However, the results obtained from difference spectra and fluorescence spectra showed incomplete reversibility with hysteresis.

This ostensible dichotomy concerning the structural thermostability of the rod portion of myosin molecule may mean that although ORD and CD studies show almost full reversibility of the helix-coil transition, local irreversible conformational changes, involving aromatic amino acid residues take place. This fact suggests that the renahired α-rope of the myosin total rod can exhibit different properties than the native molecule under conditions where no discernible loss in helix content occurs.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Dominant mutations affecting muscle structure in Caenorhabditis elegans that map near the actin gene cluster

By examining F1 progeny of mutagenized Caenorhabditis elegans larvae, we recovered several dominant mutations which affect muscle structure. Five of these new mutations resulted in phenotypes unlike the previously recognized unc-54 and unc-15 dominant alleles. Mapping studies placed all five mutations in the same small region of linkage group V. Polarized light, fluorescence and electron microscopic studies showed that a prominent feature of the disorganized myofilament lattice is the abnormal placement of thin filaments within the body wall muscle cells. Pharyngeal musculature is also affected by three of the mutations when homozygous. Of the five mutations only three are homozygous viable. All three of these have unusually high intragenic reversion rates either spontaneously (~10^?6) or after ethyl methanesulfonate mutagenesis (2 × 10^?5), suggesting that reversion occurs through loss of function mutations. No unlinked suppressor mutations were found. The dominance of the mutations, the effect on thin filaments and the reversion properties suggested that these new dominant mutations lie in a gene or genes specifying a structural component of the thin filament. The positioning of a set of three actin sequences in the same region (Files et al., 1983) led us to speculate that these mutations lie in actin genes.     

Comparison of nuclear and polysomal RNA fractions from goat brain

Phenol extracted RNA preparations from highly purified nuclei and polysomes of goat brain were fractionated by chromatography on oligo (dT)-cellulose and analyzed by electrophoresis on agarose-acrylamide composite gels. The electrophoretic profile of the polysomal polyadenylated RNA fraction showed a major band with a molecular weight of about 0.62 × 10⁶, which corresponds to the size of the tubulin mRNA. The nuclear polyadenylated RNA fraction also displayed a single major band, with an estimated molecular weight of 0.76 × 10⁶, which appears to be a potential precursor of tubulin mRNA.     

IDENTIFICATION OF A MYOSIN-LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

A myosin-like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime-mold myosin recognized a 180,000 M_r protein in western blots of total protein extracts from three different strains, including cyt-1, a cytokinesis-defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy-chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy-chain gene and a myosin-like protein in vegetative cells of the model organism Chlamydomonas.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Prenyltransferase from Gossypium hirsutum

A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophosphate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 × 10⁴ to 1.3 × 10⁵. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum.     

Identification of Phorbol Myristate Acetate Stimulated Kinase in Zea mays

Following DEAE-Sephacel and affinity chromatography a highly enriched lipid stimulated kinase activity could be recovered with a purification fold of 1725. The peak kinase activity fraction eluted with 0.1 mM calcium from phosphatidyl serine affinity chromatography showed a major protein of 70 kD and a minor band of 55 kD molecular weight and showed kinase activity that was stimulated by phorbol myristate acetate in the presence of phosphatidylserine and calcium. The optimum requirement was 2.5 × 10^?6 M, 1.25 × 10^?4 M, 1 × 10^?4 M, and 1.7 × 10^?6 M for phorbol myristate acetate, phosphatidyl serine, oleyl acetyl glycerol and free calcium respectively. The kinase activity was inhibited by H-7 and staurosporine. The binding of [³H]-phorbol myristate acetate was associated with purified fraction as resolved by get electrophoresis and the kinase activity was also precipitated by animal protein kinase C antibodies. The present data give strong evidence for the presence of phorbol myristate acetate stimulated kinase in plants.     

The structure of high molecular weight dextrins obtained from potato starch by treatment with Bacillus macerans enzyme

Bacillus macerans enzyme (BME)-derived high molecular weight dextrins, which are by-products in the course of the industrial production of cylodextrins, were isolated and their chemical structures were characterized.Dextrin I was obtained in a yield of about 24% from BME-hydrolyzate (a mixture of dextrin and cylodextrins, 50% each) of potato starch by fractionation with an ultrafiltrator having a membrane of cut-off molecular weight 2.0 × 10⁴. Dextrin II was obtained in a yield of about 15% from BME-hydrolyzate (a mixture of dextrins and cyclodextrins, 70 : 30) of Dextrin I by the same method.Dextrin I and II consisted of dextrin having molecular weights over 20 × 10⁶ and dextrins having molecular weights 4 × 10³−1 × 10⁵ in the ratio of 80 : 12 and 66: 15, respectively.The results of hydrolysis by β-amylase and methylation analysis indicated that the average, exterior and interior chain lenghts of the dextrins having molecular weights over 20 × 10⁶ and 4 × 10³−1 × 10⁵ from Dextrin I were 16.5, 8.2 and 7.3, and 11.5, 6.9 and 3.6, respectively, than those from Dextrin II were 13.6, 4.7 and 9.9, and 10.4, 5.1 and 4.3, respectively.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.     

Mechanism of reversible glycine cleavage reaction in Arthrobacter globiformis. Function of lipoic acid in the cleavage and synthesis of blycine.

The physical and chemical characterization of horse serum butyrylcholinesterase has been extended. The results show that the enzyme is a glycoprotein containing about 20% carbohydrate by weight. Mannose, glucosamine, galactose, and sialic acid are the sugar residues found. The extinction coefficient of butyrylcholinesterase, E_1cm^1% at 280 nm, was found to be 15.2 ± 0.3 by dry weight determination. The molecular weight of the protein in dilute phosphate buffer was determined to be (31.7 ± 1.2) × 10⁴ by high speed equilibrium sedimentation with a redetermined partial specific volume of 0.723 ± 0.003 ml/g. Subunit molecular weights for the dissociated protein were found to be (7.9 ± 0.4) × 10⁴ and (8.1 ± 0.1) × 10⁴, respectively, in guanidine hydrochloride and in a solution at pH 11.8. The subunit molecular weight was also estimated to be (8.8 ± 0.2) × 10⁴ by analytical sodium dodecyl sulfate-gel electrophoresis. This apparently higher subunit molecular weight from dodecyl sulfate gels is expected for glycoproteins containing significant amounts of carbohydrate. No free sulfhydryl group was detected, even though there are six half-cystines in each subunit. Therefore, it seems likely that there are three pairs of disulfide bonds per subunit. The available data indicate that native butyrylcholinesterase is a tetrameric glycoprotein consisting of subunits of equal molecular weight.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.