Effect of norepinephrine on chromatin protein phosphorylation in C-6 glioma cells

Rat C₆ glioma cultures were exposed to labelled sodium phosphate after treatment with NE with or without propanolol. Histones and non-histone proteins (NHP) were extracted from chromatin and there was no significant change in the specific activity of the total pool of histones and NHP between control and other two groups. However, after electrophoretic separation F₂a₂ histone showed a 60% increase while F₂b and F₃ histones exhibited a 40% decrease in phosphorylation in response to NE. There was no significant change in the gel pattern of NHP from different groups on SDS-PAGE. However, the 30k dalton NHP showed an increase in phosphorylation in response to NE and this increment was blocked by propanolol. The possible role of β-receptors on nuclear protein phosphorylation and genomic expression is discussed.     

Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F₁ was most evident when the lysing medium contained 1 mm Mg²⁺ and Ca²⁺ and 5 mm citrate (pH 2.75). In these conditions twice as much F₁ is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 mm Mg²⁺ and Ca²⁺ and no citrate. In these conditions more F_{2a 1} is retained in exponential than in stationary nuclei while the opposite is true for F₃. However, the total amount of arginine-rich fractions (F_{2a 1} + F₃) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.     

Histones from exponential and stationary L-cells. Evidence for metabolic heterogeneity of histone fractions retained after isolation of nuclei

Histones of exponential and G₁-arrested mouse L-cells were double-labeled with either [³H]lysine and [³²P]phosphate or with [¹⁴C]arginine and [³H]acetate in order to investigate cell cycle-dependent changes in rates of synthesis and metabolic modifications as well as the effect of the method of nuclear isolation on retention of the labeled fractions. Results indicate that newly synthesized lysine-rich histones incorporate ³²P at the highest rate. However, phosphorylation can also be detected in G₁-arrested cells and in the case of F_2b, F_1⁰, and F_1¹, with no detectable new synthesis.On the other hand, acetylation proceeded at similar rates in both exponential and stationary cells with the exception of F_{2a 2} and F₃ which showed higher acetylation levels in the latter.In relation to the method used for nuclear isolation, we observed that, even in cases where no difference in the relative amount of a given histone could be detected, the species recovered were not identical. We conclude that none of the methods commonly employed retains all of the histones. In general, the acetylated species appear to be best preserved at a higher divalent cation concentration while the newly synthesized ones are better conserved at lower ionic strengths.     

Relationships of Histones to RNA Synthesis in Plant Tissues

The RNA-synthesizing activity of the tissue, template activity of the chromatin, histones and other parameters were analyzed for young leaves, senescent leaves and the pith tissue of tobacco. The amount, of RNA, DNA and the extent of incorporation of ³²P into RNA was much lower in old leaves and the pith tissue than in young leaves. Furthermore, the ³²P sucrose density gradient patterns of RNA from the three tissues were very different. In old leaves, the label was found mostly in low molecular weight RNA region, presumably as a result of degradation of RNA by soluble and chromatin-associated ribonucleases which were higher in old leaves. — In addition to significant differences in the composition of chromatin, large differences in the ratios of F_I : F_II : F_III : : histone fractions from the three tissues were noted and the fully differentiated old leaves and the pith tissue had proportionately more F_IIand F_III histones than the less differentiated young leaves. The F_III histone of tobacco differed from that of pea and calf thymus in having lys/arg = 1.2. — Although some correlation between RNA-synthesizing activity of the tissue, template activity of chromatin and the histone composition was noted for the pith tissue and the young leaves, the situation with old leaves was more complicated, probably due to the occurrence of chromatin-associated deoxyribonuclease and involvement of other factors which may also effect RNA synthesis.     

Stimulation of minor fetal hemoglobin synthesis in cord blood reticulocytes by butyrate

As it had been shown that sodium butyrate promoted hyperacetylation of nuclear histones the effect of 5, 10, and 50 mM sodium butyrate on the $\text{in}\text{vitro}$ synthesis of Hb F_Ic (an acetylated component of Hb F) and Hb F in cord blood reticulocytes was studied. The presence of 5 mM butyrate showed a significant increase in the production of Hb F_Ic, whereas Hb F synthesis was unaffected. Higher concentrations of butyrate, however, had no stimulatory effect on Hb F_Ic synthesis. Results of pulse-chase studies indicated that sodium butyrate inhibited the turnover of preformed Hb F_Ic, probably by inhibiting deacetylase.     

Die Interfertilitätsbeziehungen europäischer Sippen derArabis hirsuta-Gruppe (Brassicaceae)

Intraspecific hybrids are always fertile. In interspecific crosses total or nearly total seed sterility regularly occurs in F₁ or F₂ individuals or even in the involved pods of P generations: Sterility barriers range from F₁ failure to moderate reduction of average hybrid fertility, and are reflected by the taxonomy of the group. Diploid F₁ hybrids are able to produce triploid descendants, triploid F₁ plants however pentaploid to hexaploid ones. Intermediary and dominant inheritance of indument characters and new character combinations in F₂ generations are observed.     

The falcifolia syndrome of Oenothera: III. The general pattern of its non-Mendelian inheritance

Summary The falcifolia (fal) syndrome is a malformation characterized by shoot sectors with sickle-shaped leaves, which appears in hybrids between Oenothera suaveolens and O. lamarckiana and shows a non-chromosomal inheritance of a previously undescribed type. The determinants, their location in the cell, and the mechanism of their expression are unknown. The defect is the result of a cross in which mixing of two different cytoplasms occurs, without the usual predominantly maternal inheritance. F₁ progeny of reciprocal crosses show a quantitative difference in the frequency and degree of expression of the fal character. When the F₁ progeny are backcrossed to the parents, the percentage of fal is high in crosses to O. suaveolens and low in those to O. lamarckiana. This manner of transmission is observed regardless of whether the hybrid is used as seed or pollen parent or shows a normal or fal phenotype. F₂ generations from F₁ plants having either a normal or a fal phenotype always include a certain percentage of fal plants, although the latter generally produce a higher percentage of fal progeny. If a second backcross is carried out, plants that produce normal progeny on self-pollination behave differently from those that produce some fal off-spring when selfed. The latter are similar to the F₁ with regard to the transmission of the fal trait. Plants of the F₁B₁ yielding normal progeny upon selfing produce normal progeny in the F₁B₂ if the parent to which they are backcrossed is the same as in the first backcross; if the parents of the first and second backcross differ, a high percentage of fal offspring is obtained. Again, whether the hybrid is used as seed or pollen parent is not relevant. Exceptions to this behaviour have been observed only rarely in that the character of the penultimate cross is retained. There is some evidence of somatic segregation of the fal determinants, since sister plants may react differently; this suggestion is supported by comparing the progenies of different branches of a self-pollinated fal plant of the F₁ generation.Abbreviations F₁, F₂, F₃, F₄ First through fourth filial generation, obtained by self-pollination - F₁B₁ First backcross generation, i.e. the F₁ was backcrossed to one of the original parents - F₁B₂ Second backcross generation, i.e. the F₁B₁ was backcrossed to one of the original parents - F₁B₃ Third backcross generation, i.e. the F₁B₂ was backcrossed to one of the original parents - (F₁B₁)D₁ Descendants obtained by self-pollination of a F₁B₁ plant; further generations obtained by self-pollination are designated as D₂, D₃, D₄ - (F₁B₁)D₁B₁ Descendant or generation obtained by a backcross of the D₁ of an F₁B₁. Backcrosses of the D₂ and D₃ are designated mutatis mutandis - (F₁B₁)D₁B₂ Generation obtained by a backcross of the (F₁B₁)D₁B₁     

Nucleosomes and subnucleosomes: heterogeneity and composition

Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN₁) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN₂) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN₃) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN₃ particles are rapidly converted by nuclease into mononucleosomes MN₁ and MN₂ There exists, however, a relatively nuclease-resistant subpopulation of the MN₃ mononucleosomes. These 200 bp MN₁ particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN₃ particles and the smaller mononucleosomes MN₁ and MN₂.There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN₁ is a set of naked double-stranded DNA fragments ～20 bp long. The SN₂ is a complex of a specific basic nonhistone protein (molecular weight ～16,000 daltons) and a DNA fragment ～27 bp long. The SN₃ contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ～27 bp long. The SN₄ contains histones H2a, H2b, H4 and a DNA fragment ～45 bp long. The SN₅ contains histones H2a, H2b, H3 and a DNA fragment ～55 bp long. The SN₆ is a complex of histone H1 and a DNA fragment ～35 bp long. Subnucleosomes SN₇ and SN₈ each contain all the histones except H1, and DNA fragments ～100 and ～120 bp long, respectively.Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.     

Optimization of multienzyme production by two mixed strains in solid-state fermentation

F₃ and F₄ strains of Aspergillus niger were screened from five strains of fungi to produce multienzyme preparations (containing cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase) as dietary supplementation. Enzyme activities indicated that 1:4 (F₃ to F₄) was the optimum mixture proportion, and 0.3% (W/W) was the preferable pitching rate. In bran mash containing 54.5% (W/W) water, F₃ and F₄ could produce the supplementation better when cultured 30 to 36 h at 30 °C. Monofactorial and orthogonal experiments were performed to optimize media. Results of the variance and range analysis showed that the optimum medium contained 80 g of bran, 20 g of cottonseed powder, 1 g of (NH₄)₂SO₄, and 0.1 g of KH₂PO₄. When F₃ and F₄ strains were cultured in the optimum medium containing 54.5% (W/W) water, the activity of cellulase, hemicellulase, glucoamylase, pectinase, and acidic proteinase reached 996; 15,863; 13,378; 7,621; and 5,583 U/g, respectively.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

Genetic evidence for gametophytic selection of wilt resistant alleles in chickpea

Gametophytic selection can drastically reduce the number of selection cycles during crop improvement programs. The objective of the present investigation was to test whether the nature of inheritance of two unlinked disease-resistant loci, h ₁ and h ₂, against Fusarium wilt in chickpea (Cicer arietinum L.) under gametophytic (pollen) selection was similar to that already observed at sporophytic level. A homozygous dominant (H ₁ H ₁ H ₂ H ₂) susceptible genotype JG-62 was crossed to a recessive (h ₁ h ₁ h ₂ h ₂) resistant genotype WR-315 to produce 20 F₁ hybrid seeds. In the following generation, flower buds of 10 F₁ hybrid plants were subjected to toxin stress before anthesis and the remaining ten control F₁ plants’ flowers were sprayed with water. Thirty-four selected BC₁ plants were generated by test crossing resistant WR-315 individuals with pollen from toxin-stressed F₁ individuals. Both control and treated F₁ plants were selfed to produce respective F₂ generations. Two DNA markers, CS-27_700bp and A07C_430bp, linked to susceptible alleles H ₁ and H ₂, respectively, were used to study the inheritance patterns of h ₁ and h ₂ loci in the F₂ and BC₁ generations. One hundred and forty-four selected F₂, 129 control F₂, and 34 selected backcross individuals were tested for the presence or absence of DNA markers. Except for the control F₂, observed ratios of selected F₂ and BC₁ populations exhibited significant chi-square deviations from expected monogenic and digenic ratios. Our results suggest that gametophytic selection is as effective as that realized at the sporophytic level, and that the gametophytic selection can be an effective breeding tool for plant breeding programs.     

F₂-isoprostane formation, measurement and interpretation: the role of exercise

The level of F₂-isoprostanes (F₂-IsoP) in blood or urine is widely regarded as the reference marker for the assessment of oxidative stress. As a result, nowadays, F₂-IsoP is the most frequently measured oxidative stress marker. Nevertheless, determining F₂-IsoP is a challenging task and the measurement is neither free of mishaps nor straightforward. This review presents for the first time the effect of acute and chronic exercise on F₂-IsoP levels in plasma, urine and skeletal muscle, placing emphasis on the origin, the methodological caveats and the interpretation of F₂-IsoP alterations. From data analysis, the following effects of exercise have emerged: (i) acute exercise clearly increases F₂-IsoP levels in plasma and this effect is generally short-lived, (ii) acute exercise and increased contractile activity markedly increase F₂-IsoP levels in skeletal muscle, (iii) chronic exercise exhibits trend for decreased F₂-IsoP levels in urine but further research is needed. Theoretically, it seems that significant amounts of F₂-IsoP can be produced not only from phospholipids but from neutral lipids as well. The origin of F₂-IsoP detected in plasma and urine (as done by almost all studies in humans) remains controversial, as a multitude of tissues (including skeletal muscle and plasma) can independently produce F₂-IsoP.     

Structural characterization of sulfated arabinans extracted from Cladophora glomerata Kützing and their macrophage activation

Water-soluble sulfated heteropolysaccharides were extracted from Cladophora glomerata Kützing and fractionated by ion-exchange chromatography, which yielded two subfractions, F₁ and F₂. The crude and fractionated polysaccharides (F₁ and F₂) mostly consisted of carbohydrates (62.8–74.5%) with various amounts of proteins (9.00–17.3%) and sulfates (16.5–23.5%), including different levels of arabinose (41.7–54.4%), galactose (13.5–39.0%), glucose (0.80–10.6%), xylose (6.84–13.4%), and rhamnose (0.20–2.83%). Based on the size exclusion chromatography (SEC) profiles, the crude and fractions mainly contained one peak with shoulders having molecular weight (M_w) ranges of 358–1,501 × 10³. The F₁ fraction stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines compared to the crude and F₂ fraction. The backbone of the most potent immunostimulating fraction (F₁) was α-(1→4)-L-arabinopyranoside with galactose and xylose residues as branches at O-2 position, and sulfates mainly at O-2 position as well.     

In vitro acylation of the ϵ-amino group of l-lysine in calf thymus histones by the carcinogen, β-propiolactone

We had previously reported that the carcinogen, β-propiolactone (BPL) reacted in vitro with histones in whole mouse skin chromatin and that among the histone classes BPL was preferentially bound to the lysine-rich histones H1 and H1°. In order to determine if in vitro reaction of BPL with calf thymus histones resulted in binding of BPL to l-lysine, we synthesized the model compounds ?-N-(3-hydroxypropionyl)lysine (HPL) and ?-N-(2-carboxyethyl)lysine (CEL) from BPL and l-lysine. The α-amino group of l-lysine was protected from reaction with BPL by the formation of a copper chelate.Structures were assigned on the basis of infrared spectra, pKa values and chemical analyses. BPL was reacted in vitro with calf thymus histones and the BPL-reacted calf thymus histones and control calf thymus histones were digested with trypsin followed by pronase. The respective digests were each chromatographed on a column of AA-15 cation-exchange resin. The elution profiles of the two digests were very similar except for the appearance of a new ninhydrin-positive peak (NNPP) in the eluate of the trypsin-pronase digest of BPL-reacted calf thymus histones. When compounds HPL and CEL were added to the trypsin-pronase digest of control calf thymus histones and the mixture chromatographed on AA-15, both compounds were resolved from the other peptide (or amino acid) peaks. HPL was eluted in the same fractions as NNPP, HPL and NNPP exhibited identical R_F values on silica gel TLC with acidic, alkaline and neutral solvents. CEL was not identified as a product of the reaction between BPL and calf thymus histones.     

Isolation,Identification and Some Cultural Conditions of Streptomyces Species That Produce Water-insoluble Polyglucan Hydrolase

Cariogenic streptococci produce tenacious water-insoluble polysaccharides from sucrose and these form the structural intercellular matrix of dental plaque. Two Streptomyces species were isolated from soils on agar medium containing the water-insoluble polyglucan as a sole carbon source. They were identified as Streptomyces werraensis (strain F₁) and Streptomyces chartreusis (strain F₂). These strains produced extracellular enzymes which strongly solubilized the polyglucans from various strains of cariogenic streptococci. Strain F₂ produced polyglucanases under rather stationary cultural condition, while F₁ required vigorous aeration. The polyglucanases may provide a useful measure for the prevention and control of dental plaque formation,     

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD₅₀=1.5 μM). Extracellular Aβ precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD₅₀=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD₅₀=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated Aβ precursor. Cells expressing the Aβ precursor-directed ribozyme bound less ¹²⁵I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface Aβ precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.     

Genetic Analysis of Tolerance to Rice Tungro Bacilliform Virus in Rice (Oryza sativa L.) Through Agroinoculation

Balimau Putih [an Indonesian cultivar tolerant to rice tungro bacilliform virus (RTBV)] was crossed with IR64 (RTBV, susceptible variety) to produce the three filial generations F₁, F₂ and F₃. Agroinoculation was used to introduce RTBV into the test plants. RTBV tolerance was based on the RTBV level in plants by analysis of coat protein using enzyme‐linked immunosorbent assay. The level of RTBV in cv. Balimau Putih was significantly lower than that of IR64 and the susceptible control, Taichung Native 1. Mean RTBV levels of the F₁, F₂ and F₃ populations were comparable with one another and with the average of the parents. Results indicate that there was no dominance and an additive gene action may control the expression of tolerance to RTBV. Tolerance based on the level of RTBV coat protein was highly heritable (0.67) as estimated using the mean values of F₃ lines, suggesting that selection for tolerance to RTBV can be performed in the early selfing generations using the technique employed in this study. The RTBV level had a negative correlation with plant height, but positive relationship with disease index value.     

Additive and additive x additive interaction make important contributions to spikelets per panicle in rice near isogenic （Oryza sativa L.） lines

Epistasis plays an important role in the genetic basis of rice yield traits. Taking interactions into account in breeding programs will help the development of high-yielding rice varieties. In this study, three sets of near isogenic lines (NILs) targeting three QTLs for spikelets per panicle (SPP), namely qSPP1, qSPP2 and qSPP7, which share the same Zhenshan 97 genetic background, were used to produce an F₂ population in which the three QTLs segregated simultaneously. The genotypes of the individual F₂ plants at the three QTLs were replaced with three markers that are closely linked to the corresponding QTLs. These QTLs were validated in the F₂ and F₃ populations at the single marker level. qSPP7 exhibited major pleiotropic effects on SPP, plant height and heading date. Multifactor analysis of variance was performed for the F₂ population and its progeny. Additive (additive interaction between qSPP2 and qSPP7 had significant effects on SPP in both the F₂ population and its progeny. Both additive and additive (additive interactions could explain about 73% of the total SPP phenotypic variance. The SPP performance of 27 three-locus combinations was ranked and favorable combinations were recommended for rice breeding in different ecosystems.     

GISH study of advanced generation of the interspecific hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew

Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F₂ hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their complements 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F₅ hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC₁F₅ hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC₂ (BC₁F₅) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed.     

The segregation pattern of a translocation quadrivalent

The segregation pattern of a translocation quadrivalent was studied in three hybrid families of the tetraploid (2n=28) oat, of the Avena strigosa polyploid complex. The rate of IV formation in F₁ was high and the fertility was normal. The adjacent-alternate orientation of this quadrivalent was very variable. Conspicuous variation in this frequency was found between anthers of the same floret and between florets. The alternate type was usually more common toward the end of MI. The adjacent-alternate ratio was found to be an unreliable measure for calculating the type of gametes produced by the F₁ and the F₂ plants derived from them. An attempt was made to determine the type of viable gametes produced on the F₁ and their frequency by examining the cytology of F₂ plants. The various combinations of the chromosomes of the translocation complex expected in the F₂ plants were derived and their expected frequencies calculated by taking into account chiasma formation at the chromosome ends and various restrictions of gamete viability. In none of the three hybrid families F₂ individuals were found to produce trivalents as the most complex chromosome configuration, indicating that one type of gamete derived from adjacent separation was not formed. The 11 ratio between F₂ plants having only bivalents (2II) and those with F ₁-like quadrivalent configuration R(c), expected when gametes resulting from alternate separation are fully functional, with the exclusion of other types, was not found. That ratio 2II/R(c), was 2-1/3 in the various families. The cytology of selected F₃ individuals basically followed the predictions based on chromosome association in the F₂.