Genotyping-by-sequencing to remap QTL for type II Fusarium head blight and leaf rust resistance in a wheat–tall wheatgrass introgression recombinant inbred population

Fusarium graminearum Schwabe (Fusarium head blight, FHB) and Puccinia triticina Eriks (leaf rust) are two major fungal pathogens posing a continuous threat to the wheat crop; consequently, identifying resistance genes from various sources is always of importance to wheat breeders. We identified tightly linked single nucleotide polymorphism (SNP) markers for the FHB resistance quantitative trait locus (QTL) Qfhs.pur-7EL and the leaf rust resistance locus Lr19 using genotyping-by-sequencing (GBS) in a wheat–tall wheatgrass introgression-derived recombinant inbred line (RIL) population. One thousand and seven hundred high-confidence SNPs were used to conduct the linkage and QTL analysis. Qfhs.pur-7EL was mapped to a 2.9 cM region containing four markers within a 43.6 cM segment of wheatgrass chromosome 7el₂ that was translocated onto wheat chromosome 7DL. Lr19 from 7el₁ was mapped to a 1.21 cM region containing two markers in the same area, in repulsion. Five lines were identified with the resistance-associated SNP alleles for Qfhs.pur-7EL and Lr19 in coupling. Two SNP markers in the Qfhs.pur-7EL region were converted into PCR-based KASP markers. Investigation of the genetic characteristics of the parental lines of this RIL population indicated that they are translocation lines in two different wheat cultivar genetic backgrounds instead of 7E–7D substitution lines in Thatcher wheat background, as previously reported in the literature.     

Possible Origins of CTnBST, a Conjugative Transposon Found Recently in a Human Colonic Bacteroides Strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

The mechanism of integration of phage Mu in the chromosome of Escherichia coli.

Cultures of synchronized $\text{E. coli}$ were infected with phage Mu at various stages of the division cycle. Phage integration at a given locus on the chromosome was measured by the lost of the corresponding gene function. For several loci, maximal integration occured at gene locus during replication of that locus.     

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.     

Crystallization and preliminary x-ray investigation of ubiquitin, a non-histone chromosomal protein.

Large crystals of bovine thymus ubiquitin, a non-histone chromosomal protein, were grown from polyethylene glycol solutions. The crystals are orthorhombic, space group P2₁2₁2₁, with $\text{a = 50·9}\text{a}\text{?}\text{, b = 42·9}\text{A}\text{?}\text{and}\text{c = 29·0}\text{A}\text{?}$. The asymmetric unit contains one ubiquitin molecule.     

A 3H-labelled trisaccharide from heparin as substrate for acetyl:CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase

The tetrasaccharide fraction obtained by gel chromatography after treatment of commercially available heparin with nitrous acid was reduced with NaB³H₄ and then hydrolysed with 2m trifluoracetic acid at 70° for 3 days. By gel chromatography and electrophoresis, the ³H-labelled trisaccharide 1 bearing an unsubstituted 2-amino-2-deoxy-d-glucosyl group in the non-reducing position was obtained (18% from the ³H-labelled tetrasaccharide). By sequential, enzymic degradation, the structure $\text{α-}\text{d}\text{-GlcN}\text{-(1→4)-β-}\text{d}\text{-GlcA}\text{-(1→4)-[1-}{}^3\text{H]aManol}$ was obtained for 1, which is a substrate for acetyl-CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, an enzyme that is deficient in the Sanfilippo C syndrome. In human-skin fibroblasts, the pH optimum of acetyl transfer onto 1 was between pH 5.5 and 7.0, and dependent on the buffer. An apparent K_m for 1 of 0.14mM was found.     

Effect of <Emphasis Type="Italic">rad50</Emphasis> mutation on illegitimate recombination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura⁺ phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.     

Studies on the biosynthesis of N-(γ-L-glutamyl)-4-hydroxyaniline] in Agaricus bisporus: Identification of the position in shikimic acid at which the amination occurs

Labelled shikimic acid was efficiently incorporated into the aniline moiety of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$, a characteristic aromatic compound of the common mushroom, Agaricus bisporus. Incubations with [3-³H]- and [1,6-¹⁴C]shikimic acid clearly proved that the amination of shikimic acid occurs at its 4-position during the biosynthesis of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

Isolation and characterization of Hfr males in Citrobacter freundii

Citrobacter freundii Hfr donor strains were isolated from a C. freundii strain harbouring a temperature-sensitive factor F ts 114 lac ⁺, by selecting for integrative suppression of the ts 114 mutation. Three Hfr strains were characterized, which transfer their chromosomes in a linear and oriented order. The first strain transfers: O-aro ⁺-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺, the second: O-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺ and the third: O-ilv ⁺-aro ⁺-nad ⁺-his ⁺-pro ⁺. The whole chromosome is transferred into the recipient cell within about 145 minutes. From these results we concluded that the linkage map of C. freundii is circular. Mating-pair formation on a membrane filter resulted in more recombinants being formed as compared with mating-pair formation in liquid medium. Furthermore the mating-pairs formed on a membrane were more stable. From one Hfr strain heterogenic F-prime factors could be isolated bearing the F ts 114 lac ⁺ genes from Escherichia coli and the pur ⁺ and/or ilv ⁺ genes from C. freundii. Preliminary mapping by interrupted mating indicated that the linkage map of C. freundii is in general very similar to those of E. coli, Salmonella typhimurium and Klebsiella aerogenes.     

Site of action of the crinkled (cr) locus in the mouse.

The site of action of the crinkled (cr) locus was determined by combining dermis and epidermis from the tail of 15-day $\text{+}\text{cr}$ and $\text{cr}\text{cr}$ mice and by growing the recombined skins in the testes of histocompatible mice. Since $\text{cr}\text{cr}$ mice have bald tails, the presence or absence of hair in the graft was the feature used to determine gene action. Grafts of the combinations $\text{+}\text{cr}$$\text{epidermis}\text{-}\text{+}\text{cr}$ dermis and $\text{+}\text{cr}$$\text{epidermis}\text{-}\text{cr}\text{cr}$ dermis grew hair, whereas grafts of the combinations $\text{cr}\text{cr}\text{epidermis}\text{-}\text{+}\text{cr}$ dermis and $\text{cr}\text{cr}$$\text{epidermis}\text{-}\text{cr}\text{cr}$ dermis produced no hair. It was concluded that the cr locus, at least for tail skin, is active in the epidermis.     

Requirement for the Escherichia coli ribosomal protein L11 in peptide chain termination.

Subparticles of the Escherichia coli 50 S ribosome subunit containing varying amounts of the protein L11 have been prepared. These core particles have been used to form 70 S couples containing f[³H]Met-tRNA as a substrate for the peptidyl hydrolysis reaction of in vitro termination. Studies with antibodies against L11 suggested previously that the protein was involved in this event. The peptidyl transferase of the 50 S subunit core particles containing no more than 6% of the normal complement of L11 was fully active. The 70 S couples formed from 50 S cores lacking L11 showed some decrease in their ability to bind fMet-tRNA. Ribosomes lacking the proteins $\text{L}\text{7}\text{L}\text{12}$ retained about 50% of their activity for the peptidyl-tRNA hydrolysis event of in vitro termination. Cores lacking both $\text{L}\text{7}\text{L}\text{12}$ and L11 were almost as active as those lacking only $\text{L}\text{7}\text{L}\text{12}$. L11 is, therefore, not absolutely required for peptidyl-tRNA hydrolysis at termination in vitro. The ribosome subparticles lacking L11 have been reconstituted with $\text{L}\text{7}\text{L}\text{12}$. Despite the absence of L11, they regained significant activity for the codon-directed in vitro termination reaction.     

Membrane-associated reactions in ubiquinone biosynthesis: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. $\text{S-}\text{Adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-l-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O- methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal acitivity in vitro. The methyltransferase was absent from extracts from ubiG^? mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.     

An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.     

Chromosomal assignment of porcine pregnancy-associated glycoprotein gene family

This study presents the chromosomal assignment of a multiple pregnancy-associated glycoprotein (PAG) gene family in the domestic pig (pPAG). The pPAG locus was identified by physical mapping (fluorescent in situ hybridisation—FISH; with various probes), and additionally confirmed by Southern hybridisation of pPAG amplicons using laser microdissected Sus scrofa chromosome 1 (SSC1), as genomic templates. Various pPAG probes were produced with the use of diverse identified templates: pPAG1-6, -8, -10 cDNAs (GenBank: L34360–1, AF315377, AF272734, AY188554, AF272735, AY373029 and AY775784, respectively), or genomic DNA (gDNA) probes of pPAG2 gene and its promoter (GenBank: U39198–9, U39762–3, U41421–4). All probes, including long gDNA probes (～9.2 kbp GpPAG2 gene; ～2.8 kbp GpPAG2 promoter), a shorter cDNA probe (PlpPAG4, 1385 bp) and amplified pPAG2-like probes (ApPAG2L) specific for cDNA inserts of pPAG2-like gene subfamily (pPAG2, -4, -6, -8 and -10; 1283–1385 bp) were produced by random priming using biotin-labelled deoxynucleotides (16-dUTP). Numerous FISH mappings with various pPAG probes revealed the chromosomal assignment of the pPAG gene family to the long arm of porcine chromosome 1 (SSC1q16–q24 region). This cytogenetic assignment was confirmed by Southern hybridisation (with ³²P-labelled pPAG10 probe) of multiple distinct pPAG amplicons (603–3943 bp) produced with the use of 25 laser microdissected SSC1, as gDNA templates. This is the first study identifying the chromosomal locus of the pPAG gene family in the pig.     

Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of ϵ-ATP, ϵ-ADP and ϵ-AMPPNP detected by polarized fluorescence

The polarized fluorescence from nucleotides bound to myosin heads in glycerinated muscle fibers of rabbit psoas was measured as the number of myosin heads with bound nucleotides was varied by adding various concentrations of fluorescent ?-ATP, ?-ADP and ?-AMPPNP (1:N⁶-etheno-ATP, -ADP and -imido ATP). The angles of the absorption and emission dipoles of bound nucleotides to the fiber axis and their angular distribution were determined from the observed values of four components of the polarized fluorescence.The maximum amount of nucleotides bound to the myosin heads in the fiber, B_m, was 170 to 270 μm. The dissociation constant of nucleotides, $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, increased in the order ?-ATP, ?-ADP, ?-AMPPNP, and was four to six times larger at a sarcomere length (SL) of 2.1 μm than at 3.7 μm.The polarized fluorescence from bound ?-ADP at SL = 2.1 μm was independent of the amount of bound ?-ADP when it was lower than one-half of B_m, indicating a single helical array of myosin heads having ?-ADP. The angles of the absorption dipole, φ_A, and the emission dipole, φ_E, to the fiber axis were 69 ° and 66 °, respectively. As the amount of bound ?-ADP exceeded one-half of B_m, the values of the polarized fluorescence showed that the extra ?-ADP bound to myosin heads with a lower affinity and had different angles to the fiber axis: φ_A and φ_E were 49 ° and 54 °, respectively. The half-maximum width of the angular distribution of these bound ?-ADP molecules, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, was about 20 °.During development of isometric tension in the presence of ?-ATP with Mg²⁺, the polarized fluorescence was independent of the amount of bound ?-ATP when it was lower than one-third of B_m or when the concentration of free ?-ATP was lower than 100 μm, indicating a single helical array of myosin heads undergoing the ATPase reaction. The angles of bound nucleotides, φ_A and φ_E, were 68 ° and 64 °, respectively. The half-maximum width of the angular distribution, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, was about 22 °. As the amount of bound nucleotides exceeded one-third of B_m, the polarized fluorescence showed deviation from the values expected for the single helical array.The angles φ_A and φ_E for bound ?-AMPPNP were about 58 ° and 62 °, respectively, but the angular distribution was broad; that is, $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was about 42 °. These angles were independent of the amount of bound ?-AMPPNP.In a stretched fiber with SL = 3.7 μm, the polarized fluorescence showed that the angles of ?-ADP, ?-ATP and ?-AMPPNP bound to myosin heads had almost random distributions; $\text{θ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 90 ° to 100 °, independent of the amount of bound nucleotides. Similar results were obtained with the relaxed fiber in the presence of ?-ATP.     

Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene.

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M·Eco P1) or with the DNA-adenine methylase (M·Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M·Eco dam modification of calf thymus DNA. The results show that M·Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M·Eco P1 has also been deduced; we propose that M·Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A¹-C-Py … 3′ (where $\text{A}{}^1\text{= N}{}^6$ methyladenine and Py is C or T).     

Engineering Corynebacterium glutamicum with a comprehensive genomic library and phage-based vectors

The Gram-positive bacterium Corynebacterium glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. glutamicum BT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. glutamicum as a leading host for drug discovery.     

Reactions of chitosan: 5. The reaction of chitosan with 2,4-dinitrofluorobenzene and determination of the extent of the reaction

The reaction between chitosan and 2,4-dinitrofluorobenzene has been studied and suitable conditions established for hydrolysis of the product prior to determination of the extent of reaction by u.v./visible spectroscopy. The chromophore system in $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$, the final product from the acid hydrolysis of N-(2,4-dinitrophenyl)chitosan, is unstable to heating in solution in either water or aqueous acid. The temperature of hydrolysis should therefore not exceed 50°C and at this temperature the u.v./visible absorption spectrum of $\text{N-(2,4-}\text{dinitrophenyl}\text{)-2-}\text{amino}\text{-2-}\text{deoxy-}\text{d}\text{-glucose}$ is constant for up to 50 h. Complete reaction of the amine groups is not achieved under heterogeneous or homogeneous conditions, only approximately 50% of the available amine groups undergoing reaction under homogeneous conditions. This restricted reactivity results from the bulky N-(2,4-dinitrophenyl) residues shielding adjacent unreacted amine groups on the same chain, thereby preventing their reaction with 2,4-dinitrofluorobenzene. Such intramolecular steric hindrance would be expected to increase with increase in the free amine group content of the sample, due to the increase in the fraction of amine groups occurring in sequence length of two or more, and an inverse relationship between the total initial free amine group content and the percentage of these that react with 2,4-dinitrofluorobenzene has been found