Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.     

Tissue-specific position effects on alcohol dehydrogenase expression in Drosophila melanogaster

Summary Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three proteases (PrtA, PrtB and PrtC) into the extracellular medium. The gene encoding the 50 kDa protease, prtA, was subcloned from a recombinant cosmid carrying a fragment of the E. chrysanthemi B374 chromosome. prtA was shown to be located immediately 3 to the structural genes for the other two extracellular proteases. The amino acid sequence of PrtA, as predicted from the prtA nucleotide sequence, showed a high level of homology with a family of metalloproteases that are all secreted via a signal peptide-independent pathway, including PrtB and PrtC of E. chrysanthemi B374, PrtC of E. chrysanthemi EC16, PrtSM of Serratia marcescens and AprA of Pseudomonas aeruginosa. PrtA secretion requires the E. chrysanthemi protease secretion factors PrtD, PrtE and PrtF. The secretion signal of PrtA is near to the carboxy-terminal end of the protein, as was previously shown to be the case for PrtB and PrtSM and for Escherichia coli -hemolysin. The C-termini of these four proteins do not show extensive primary sequence homology, but PrtA, PrtB and PrtSM each have a potential amphipathic -helix located close to the C-terminus.     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A -glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both -glucosidase and -xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.     

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon

Summary A transducing phage carrying glpDlacZ, glpR, and malT was isolated from a strain harboring a glpDlacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpDlacZ and glpRlacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed     

Silicon carbide fiber-mediated stable transformation of plant cells

Summary Maize (Zea mays, cv Black Mexican Sweet) (BMS) and tobacco (Nicotiana tabacum, cv Xanthi) tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA. Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding -glucuronidase (GUS). Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTII) and GUS from the respective plasmids. Thirty-four BASTA-resistant BMS colonies and 23 kanamycin-resistant tobacco colonies recovered following selection contained intact copies of the BAR gene and NPTII genes, respectively, as determined by Southern blot analysis. Sixty-five percent of the resistant BMS colonies and 50% of the resistant tobacco colonies also expressed GUS activity. Intact copies of the GUS gene were observed in Southern blots of all resistant BMS and tobacco colonies that expressed GUS activity. These results indicate that a simple, inexpensive DNA delivery procedure employing silicon carbide fibers can be used to reproducibly transform cells of both monocotyledonous and dicotyledonous plant species.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitableCooperative investigation of the Minnesota Agriculture Experiment Station and the US Department of Agriculture, Agricultural Research Service. Supported in part by grants from The Quaker Oats Company, and Midwest Plant Biotechnology Consortium, USDA Subgrant # 593-0009-04. Minnesota Agricultural Experiment Station Publication No. 19,226.     

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

Summary The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca⁺⁺, Mg⁺⁺ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp ¹, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, ), , , (, ) which in the notation of Downie et al. (1981) reads atpB (E F H) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc^-) mutations. We made use of the fact that specialized transducing phages asn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn⁺, and that the resulting Asn⁺ cells grow slowly if the asn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the asn which either eliminated atp genes or affected their expression (promoter mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, is discussed.     

A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

Summary A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the -galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Mutagenic DNA repair genes on plasmids from the ''pre-antibiotic era''

Summary Resistance transfer factors are natural conjugative plasmids encoding antibiotic resistance. Some also encode mutagenic DNA repair genes giving resistance to DNA damage and induced mutagenesis. It has been shown that antibiotic resistance has been acquired by recent transposition events; however, we show here that mutagenic repair genes existed much earlier on these types of plasmids. Conjugative plasmids from eight incompatibility groups from the Murray collection of pre-antibiotic era enterobacteria were tested for complementation of mutagenic repair-deficient Escherichia coli umuC36. Although none of these plasmids carry transposon-encoded drug resistance genes, IncI1 and IncB plasmids were identified which restored ultraviolet resistance and induced mutability to umuC36 mutants. Furthermore they increased the UV resistance and induced mutability of wild-type E. coli, Klebsiella aerogenes and Citrobacter intermedius, thus showing that they could confer a general selective advantage to a variety of hosts. Like know mutagenic repair genes, complementation by these plasmid genes required the SOS response of the host cell. Nucleotide hybridisation showed that these plasmids harboured sequences similar to the impCAB locus, the mutagenic repair operon of modern-day IncI1 plasmids. The evolution of mutagenic repair genes is discussed.     

Isolation of Erwinia chrysanthemi mutants altered in pectinolytic enzyme production

Various mutations in the pectin catabolic pathway of Erwinia chrysanthemi were isolated by selection of Mu-lac insertions, resulting in expression of the lac genes inducible by pectin degradation products. This approach allowed us to isolate lacZ fusions with the genes pelC, pelD, ogl and pem, encoding pectate lyases PLc and PLd, oligogalacturonate lyase and pectin methytesterase, respectively. Moreover, we obtained mutations affecting the regulation of pectinolytic enzymes; a locus called peel appeared to be involved in induction of pectate tyases and pectin methylesterase. A second locus, called pect, may encode an activator protein acting on pectate lyase production. Both peel and pecL expression are induced in the presence of pectic polymers. The expression of the pem gene was studied in more detail by analysis of the pem-lacZ fusions. The expression of pem appears to be controlled by the negative regulatory gene kdgR, which controls alt the genes involved in pectin degradation (pem, pel, ogl, kduD, kduf, kdgK, kdgA). This study confirmed that 2-keto-3-deoxy-gluconate is a key intermediate for the induction of the pectin catabolic pathway. The three genes pem, pelD and pecl were localized in the same region, near the ade-377 marker on the genetic map of the E. chrysanthemi strain 3937. The pem gene was located more precisely on an 18kb DNA fragment containing the pelADE cluster. However, this 18 kb DNA fragment did not complement the pecl mutation. The pecL mutations were located near the ile-2 marker on the genetic map of E. chrysanthemi strain 3937.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Gene fusions to the ptsM/pel locus of Escherichia coli

Summary We have constructed gene fusions between ptsM/pel and lacZ. These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage DNA, as well as that of other lambdoid phages such as Hy-2. Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene, or two genes within the same operon. Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli. This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose. However, we have also identified Tn10 insertion mutations that confer a Man^- phenotype but have no effect on the Pel phenotype. Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes. Both of these genes are involved in mannose uptake. This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of DNA.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum

Summary Hybrid plasmids were constructed by combining in vitro the Escherichia coli plasmid pGA22, which carries the genes determining resistance to kanamycin, tetracycline, chloramphenicol and ampicillin, with the cryptic plasmids, pCG1 and pCG2, of Corynebacterium glutamicum. The hybrid plasmids were introduced into C. glutamicum and E. coli and replicated in both hosts. They expressed all the E. coli resistance phenotypes except ampicillin resistance in C. glutamicum. The levels of antibiotic inactivating enzymes encoded on these plasmids were about four to ten times lower in C. glutamicum than in E. coli. Despite the lack of expression of ampicillin resistance, -lactamase activity was detected in C. glutamicum carrying hybrid plasmids.     

The right end of MudI(Ap,lac)

Stable derivatives of the bacteriophage MudI(Ap,lac) were used to generate operon fusions in S. typhimurium which exhibit a sectoring phenotype with respect to lacZ expression. The Lac^- to Lac⁺ conversion was shown to be the result of small deletions involving the right end of the MudI element. DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage. A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac). In addition, this model explains the observed deletion formation and the Lac^- to Lac⁺ conversion in the sectoring fusions.This paper is dedicated to our padrinos, John and Marge Ingraham, whose love of truth has served us as constant inspiration     

The ‘A’ genome donor of Eleusine coracana (L.) Gaertn. (Gramineae)

Summary In an attempt to discover A and B genome donor(s) to finger millet, Eleusine coracana, or its progenitor species, E. africana (both allotetraploid 2n=4x=36), five diploid species, E. Indica, E. Floccifolia, E. multiflora, E. tristachya and E. intermedia, were crossed to finger millet and its progenitor taxon. Crosses were successful only with E. coracana. Three combinations of triploid hybrids E. coracana x E. indica, E. coracana x E. floccifolia, and E. coracana x E. multiflora were obtained and analysed. Meiotic behaviour was perfectly normal in parental species. The regular number of 18 bivalents in E. coracana, 9 bivalents in E. indica, E. intermedia, E. tristachya and E. floccifolia and 8 bivalents in E. multiflora were invariably noticed. In E. coracana x E. indica hybrids a mean chromosome pairing of 8.84_I+8.80_II+0.03_III+0.10_IV per cell was found. About 86.5% of the cells showed the typical 9_I+9_II configuration, suggesting that E. indica (AA) is one of the diploid genome donors to cultivated species E. coracana. A mean chromosome pairing of 11.08_I+7.63_II+0.16_III+0.04_IV per cell was found in E. coracana x E. floccifolia hybrids. Two to ten bivalents and varying numbers of univalents were seen in 55% of the cells. About 45% of the cells showed the 9_I+9_II configuration. Various evidence suggests that perennial E. floccifolia is a primitive member of the A genome group of Eleusine species, and it may not be a genome donor to E. coracana. In E. coracana x E. multiflora hybrids (2n=26) mean chromosome pairing of 21.45_I+1.97_II+0.13_III+0.04_IV per cell was found. About 91% of the cells were observed to have 20–26 univalents. Only a small percentage of the cells contained bivalents or multivalents. This pairing behaviour indicates that E. multiflora lacks genomic homology with the A or B genome of E. coracana. Genomically E. multiflora is a distinct species and a genomic symbol of C is assigned to it. Identification of the B genome donor species to cultivated millet. E. coracana remains elusive.