Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica.     

Characterization of the gene conversions between the multigene family members of the yeast genome

Stanley Sawyer's gene conversion detection method, implemented in his GENECONV computer program, was used to detect and characterize the gene conversions between the multigene family members of the yeast genome. This method gave different gene conversion frequencies and size distribution for gene families with two members and multigene families with more than two members. The 69 gene conversions detected in multigene families with more than two members occur at a frequency of 7.8% gene conversion/pair of genes compared and have an average size of 173+/-220 nucleotides. Larger gene conversions are found only between more similar genes, the genes involved in gene conversions are distributed almost randomly among the 16 yeast chromosomes, and the frequency of gene conversions increases as the distance between repeated genes decreases. In contrast to previous studies, no relationship was observed between the level of expression of a gene and its involvement in gene conversions. These analyses also suggest that gene conversions might occur by different mechanisms in closely linked genes and unlinked genes. The excess of converted regions at the 3? end of unlinked genes suggests that recombination with incomplete cDNA molecules is the main mechanism responsible for gene conversions between such genes.     

Sequence comparisons of odorant receptors among tortricid moths reveal different rates of molecular evolution among family members

In insects, odorant receptors detect volatile cues involved in behaviours such as mate recognition, food location and oviposition. We have investigated the evolution of three odorant receptors from five species within the moth genera Ctenopseustis and Planotrotrix, family Tortricidae, which fall into distinct clades within the odorant receptor multigene family. One receptor is the orthologue of the co-receptor Or83b, now known as Orco (OR2), and encodes the obligate ion channel subunit of the receptor complex. In comparison, the other two receptors, OR1 and OR3, are ligand-binding receptor subunits, activated by volatile compounds produced by plants--methyl salicylate and citral, respectively. Rates of sequence evolution at non-synonymous sites were significantly higher in OR1 compared with OR2 and OR3. Within the dataset OR1 contains 109 variable amino acid positions that are distributed evenly across the entire protein including transmembrane helices, loop regions and termini, while OR2 and OR3 contain 18 and 16 variable sites, respectively. OR2 shows a high level of amino acid conservation as expected due to its essential role in odour detection; however we found unexpected differences in the rate of evolution between two ligand-binding odorant receptors, OR1 and OR3. OR3 shows high sequence conservation suggestive of a conserved role in odour reception, whereas the higher rate of evolution observed in OR1, particularly at non-synonymous sites, may be suggestive of relaxed constraint, perhaps associated with the loss of an ancestral role in sex pheromone reception.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Isolating promoters of multigene family members from the polyploid sugarcane genome by PCR-based walking in BAC DNA

The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.     

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.     

Desaturase multigene families of Brassica napus arose through genome duplication

 This paper reports the estimated gene copy number and restriction fragment length polymorphism (RFLP) map locations of five different desaturase cDNA clones from Brassica napus (oilseed rape). The desaturase enzymes encoded by four of these genes catalyze successive reactions that insert double bonds into lipid-linked fatty acid residues. Delta-12 (e2) and delta-15 (e3) desaturases are active in the endoplasmic reticulum, while omega-6 (p2) and omega-3 (p3) desaturases catalyze analogous desaturation reactions via a parallel pathway located in plastids. The fifth cDNA clone (b5) contains a desaturase-like domain bound to a cytochrome b₅ segment. Estimates of gene copy number based on Southern blot analysis of 16 oilseed rape varieties and three different resynthesized Brassica napus lines indicated that e2 had 4–6 gene copies and e3, p2, p3 and b5 each had 6–8 gene copies per haploid genome. Estimates of the gene copy number for the two progenitor species, Brassica oleracea and Brassica rapa, supported the premise that all these genes were at least duplicated or triplicated in the two progenitor species before they combined to form B. napus. RFLP mapping results showed that the e2 probe detected 4 distinct loci, the e3 probe 6 loci and p2, p3 and b5 each detected 8 loci, with pairs of loci often mapping to homoeologous regions on 2 different linkage groups. The 28 mappable loci were distributed across 12 linkage groups of the B. napus map (Parkin et al. 1995) and were usually represented by single RFLP fragments. A collinear segment containing the e2 and p3 loci was positioned on B. napus linkage groups N1, N11, N3, N13, N5 and N15. This segment was collinear with a 30-cM region of Arabidopsis thaliana chromosome 3 that contains the homologous fad2 (e2) and fad7(p3) genes. This suggests that the desaturase multigene families arose as the result of duplication of large chromosome segments rather than duplication of individual genes. Received: 14 August 1996 / Accepted: 18 October 1996     

The mouse alpha-amylase multigene family. Sequence organization of members expressed in the pancreas, salivary gland and liver

The DNAs that specify the α-amylase messenger RNAs found in the pancreas, salivary gland and liver of mouse strain A have been isolated by molecular cloning in phage λ. Amylase clones were studied by mRNA/DNA hybrid analysis in the electron microscope, restriction endonuclease site mapping and DNA sequencing. The Amy-2^a gene, which specifies pancreatic α-amylase mRNA, measures 10·1 kb from cap to polyadenylation site and is interrupted by at least 9 intervening sequences. Amy-1^a, which specifies both salivary gland and liver α-amylase mRNAs contains at least 10 introns. The distance between the cap and polyadenylation sites used in the salivary gland and the liver measures 22·9 kb and 20 kb, respectively. Introns are located at very similar, if not identical, positions within comparable regions of Amy-1^a and Amy-2^a. The first intron of Amy-1^a, which interrupts sequences specifying 5′ non-translated regions of salivary gland and liver α-amylase mRNAs, has no counterpart in Amy-2^a. Some introns exhibit considerable sequence homology, suggesting that Amy-1^a and Amy-2^a have evolved by duplication from a common split ancestor sequence. Repetitive sequence elements occur in the introns and flanking regions of these genes. Gene titration by quantitative autoradiography reveals only one copy of Amy-1^a, but two copies of Amy-2^a per haploid mouse genome. In addition to Amy-1^a and Amy-2^a, several other amylase-like DNA sequences exist in the mouse genome. No gross rearrangements of amylase DNA sequences can be detected between germline DNA and that of various mouse tissues.     

Expression of members of the Saccharomyces cerevisiae hsp70 multigene family

The hsp70 multigene family of Saccharomyces cerevisiae is a complex multigene family, composed of members exhibiting complex patterns of regulation. Expression of some members is induced after a heat shock, whereas expression of others is repressed. Some members of the family are expressed during exponential growth. One gene, SSA3, shows an unusual pattern of expression during approach to stationary phase. While most RNAs decrease in abundance, SSA3 RNA levels dramatically increase. The constitutive expression of SSA3 in cells lacking adenylate cyclase activity suggests that cAMP modulates SSA3 expression.     

The mutational load of a multigene family with uniform members

The mutational load of a multigene family with uniform members was studied by computer simulations. Two models of selection, truncation and exponential fitness, were examined, by using a simple model of gene conversion. It was found that the load is much smaller than the Haldane-Muller prediction under the truncation selection, and that it becomes approximately equal to the value calculated by the formula, nv(1-q)/(m-nq), where n is the copy number, v is the rate of detrimental mutation per gene copy, m is the truncation point in terms of the number of detrimental genes eliminated, and q is the equilibrium frequency of detrimental mutation. However the equilibrium frequency cannot be analytically obtained. For the exponential fitness model, the load is close to the Haldane-Muller value. When there is no gene conversion, the load becomes larger than the cases with conversion both for the truncation and the exponential fitness models. Thus, gene conversion or other mechanisms that are responsible for contraction-expansion of mutants on chromosomes helps eliminating deleterious mutations occurring in multigene families.     

Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein

Sporamin, the major soluble protein of the sweet potato tuberous root, is coded for by a multigene family. Fourty-nine essentially full-length sporamin cDNAs isolated from tuberous root cDNA library have been classified by cross hybridization, restriction endonuclease cleavage pattern and ribonuclease cleavage mapping. All the cDNAs fall into one of the two distinct homology groups, subfamilies A and B, which correspond to the polypeptide classes sporamin A and B, respectively. At least 5 different sequences are detected in both of the 22 sporamin A and 27 sporamin B cDNAs. Comparison of the nucleotide sequences of the coding region of three each of sporamin A and B subfamily members, four from cDNAs and two from genomic clones, indicates that intra-subfamily homologies (94 to 98%) are much higher than inter-subfamily homologies (82 to 84%), and there are deletions or insertions of one or two codons at three locations which characterize each subfamily. Large portions of base substitutions in the coding region accompany amino acid substitutions. In contrast to the coding region, most of the structural differences among the members in the 5 and 3 noncoding regions are deletions or insertions.     

Molecular evolution of the chalcone synthase multigene family in the morning glory genome

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. Thus, for example, most of the enzymatic components of plant secondary metabolism are encoded by small families of genes that originated through duplication over evolutionary time. The dynamics of gene family evolution are well illustrated by the genes that encode chalcone synthase (CHS), the first committed step in flavonoid biosynthesis. We review pertinent facts about CHS evolution in flowering plants with special reference to the morning glory genus, Ipomoea. Our review shows that new CHS genes are recruited recurrently in flowering plant evolution. Rates of nucleotide substitution are frequently accelerated in new duplicate genes, and there is clear evidence for repeated shifts in enzymatic function among duplicate copies of CHS genes. In addition, we present new data on expression patterns of CHS genes as a function of tissue and developmental stage in the common morning glory (I. purpurea). These data show extensive differentiation in gene expression among duplicate copies of CHS genes. We also show that a single mutation which blocks anthocyanin biosynthesis in the floral limb is correlated with a loss of expression of one of the six duplicate CHS genes present in the morning glory genome. This suggests that different duplicate copies of CHS have acquired specialized functional roles over the course of evolution. We conclude that recurrent gene duplication and subsequent differentiation is a major adaptive strategy in plant genome evolution.     

Identification and genetic mapping of 151 dispersed members of 16 ribosomal protein multigene families in the mouse

More than 150 individual members of 16 ribosomal protein multigene families were identified as DNA restriction fragments and genetically mapped. The ribosomal protein gene-related sequences are widely dispersed throughout the mouse genome. Map positions were determined by analysis of 144 progeny mice from both an interspecific (C57BL/6J × SPRET/Ei)F₁ × SPRET/Ei and an intersubspecific (C57BL/6J × CAST/Ei)F₁ × C57BL/6J backcross. In addition, 30 members of the multigene families encoding PGK1 ODC, and TPI, including five new loci for ODC and one new locus for TPI, were characterized and mapped. Interspecific backcross linkage data for 29 nonecotropic murine leukemia retroviruses endogenous to C57BL/6J mice are also reported. Transmission ratio distortions and recombination frequencies are compared between the two backcrosses.     

The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas.

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus that extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen-recessive S15 haplotype. Both genes are interrupted by a single intron; however, SLGA encodes both soluble and membrane-anchored forms of SLG, whereas SLGB encodes only soluble SLG proteins. Thus, including SRK, the S locus in the S15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized, and each SLG glycoform was assigned to an SLG gene. Evidence is presented that the S2 and S5 haplotypes carry only one or the other of the SLG genes, indicating either that they are redundant or that they are not required for the self-incompatibility response.     

Sequence analysis and comparison of cDNAs of the zein multigene family .

The nucleotide sequence of two zein cDNAs in hybrid plasmids A20 and B49 have been determined. The insert in A20 is 921 bp long including a 5' non-coding region of 60 nucleotides, preceded by what is believed to be an artifactual sequence of 41 nucleotides, and a 3' non-coding region of 87 nucleotides. The B49 insert is 467 bp long and includes approximately one-half the protein coding sequence as well as a 3' non-coding region of 97 nucleotides. These sequences have been compared with the previously published sequence of another zein clone, A30 . A20 and A30 , both encoding 19 000 mol. wt. zeins , have approximately 85% homology at the nucleotide level. The B49 sequence, corresponding to a 22 000 mol. wt. zein, has approximately 65% homology to either A20 or A30 . All three zeins share common features including nearly identical amino acid compositions. In addition, the tandem repeats of 20 amino acids first seen in A30 are also present in A20 and B49 .     

Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome.In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms.Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products.In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.     

Sequence of an expressed human beta-tubulin gene containing ten Alu family members

The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence.