Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.     

Structure of the multigene family of MAL loci in Saccharomyces

Summary Multigene families are a ubiquitous feature of eukaryotes; however, their presence in Saccharomyces is more limited. The MAL multigene family is comprised of five unlined loci, MAL1, MAL2, MAL3, MAL4 and MAL6, any one of which is sufficient for yeast to metabolize maltose. A cloned MAL6 locus was used as a probe to facilitate the cloning of the other four functional loci as well as two partially active alleles of MAL1. Each locus could be characterized as a cluster of three genes, MALR (regulatory), MALT (maltose transport or permease) and MALS (structural or maltase), encoded by a total of about 7 kb of DNA; however, homologous sequences at each locus extend beyond the coding regions. Our results indicate that there is extensive homology among the MAL loci, especially within their maltase genes. The greatest sequence diversity occurs in their regulatory gene regions. Southern cross analyses of the cloned MAL loci indicate a single duplication of the MAL6R-homologous sequences upstream of the MAL6R gene as well as an extensive duplication of more than 10 kb at the MAL3 locus. The large repeat at the MAL3 locus results in the presence of four copies of MAL3R-homologous sequences and two copies of MAL3T-homologous sequences at that locus. Two naturally occurring inactive alleles of MAL1 show a deletion or divergence of their MALR sequences. The significance of these repeats in the evolution of the MAL multigene family is discussed.     

Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Biochemical characterization of rice glutelin

The two major subunits of rice glutelin, the acidic (α) and basic (β) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the α polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the β polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the α and β polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the α-group and nine polypeptides of the β-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified α and β subunits were analyzed. The α subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the β subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the β subunits than the α subunits of several diverse plant species.     

Differential tissue expression of the glutathione transferase multigene family.

The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.     

不同胁迫条件下化感与非化感水稻PAL多基因家族的差异表达

水稻苯丙氨酸解氨酶(PAL)调控酚酸类化感物质的合成代谢。编码PAL的基因是一个基因家族,包含至少11个基因成员,并受不同环境条件的调控。为了明确PAL基因家族中调控水稻化感作用的特定基因成员,本研究运用实时荧光定量PCR技术分析了低氮及稗草胁迫条件下强化感水稻PI312777与非化感水稻Lemont中根系的11个PAL成员基因的表达差异。结果表明,低氮和稗草胁迫条件下,PI312777和Lemont中的 PAL4和PAL10均不表达,其余9个PAL基因成员发生了不同程度的表达变化。其中,PAL11均上调表达,其分别在低氮处理和稗草胁迫的PI312777中上调3.29倍和1.07倍,而在相同处理下的Lemont中上调3.92倍和1.08倍;PAL3和PAL9则仅在低氮和稗草胁迫条件下的PI312777中上调表达,低氮胁迫分别为1.83倍和2.66倍,稗草胁迫为1.46倍和2.65倍;而这两个基因在相同处理下的Lemont中表达下调,低氮胁迫下调1.05和1.24倍,稗草胁迫下调1.14和1.16倍,推测PAL3和PAL9可能与胁迫初期调控水稻化感作用有关。     

Extraction and composition of rice endosperm glutelin

“Crude” glutelin was prepared from milled rice (Oryza sativa) flour by sequential extraction of the albumin-globulin fraction with 0.5 M NaCl and prolamin with 70% ethanol-0.6% β-mercaptoethanol. The solvent, 0.5% sodium dodecyl sulphate (SDS)-0.6% β-mercaptoethanol, extracted 91% of the endosperm glutelin without gelatinizing starch granules, whereas chaotropic solvents such as urea and guanidine caused extensive gelatinization. The S-cyanoethyl glutelin (Ce-glutelin) prepared by SDS extraction of the “crude” glutelin (9.5% protein) of IR480-5-9 rice gave three major subunits with MW 38000, 25000 and 16000 in the ratio 2:1:1 as determined by SDS polyacrylamide gel electrophoresis. A similar preparation from “crude” glutelin of a lower protein containing rice had the corresponding subunits in the ratio of 16:3:1. The MW 38000 subunit was unique to glutelin and was not present in C3-albumin-globulin or prolamin; the subunits were only partially purified by SDS Sephadex G-150 gel-filtration. The C3-glutelin was also prepared from a crude glutelin-prolamin preparation from IR480-5-9 by NaOH extractions followed by precipitation at pH 10 and ethanol extraction of the precipitate (C3-glutelin). This preparation had the same three major subunits and in the same ratio as C3-glutelin prepared by the SDS method. The subunits of the former preparation were separated by carboxymethyl Sephadex C-50 chromatography; the MW 38000 subunit eluted between pH 6.2–8.5, the MW 25000 in an impure state at pH values above 9, and the MW 16000 subunit was eluted at pH 8.6—9.2. Amino acid composition of the Ce-glutelin preparations were similar to each other. The MW 38000 and 16000 subunits had lower lysine contents than whole C3-glutelin, whereas the MW 25000 subunit had a higher lysine content.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

Sequence of three members and expression of a new major subfamily of glutelin genes from rice

Three members have been isolated of an additional glutelin gene subfamily, named subfamily B, consisting of about five members per haploid rice genome. Restriction fragment length polymorphism analysis showed major differences between Japonica and Indica lines, indicating the divergence of the subfamily since the split between the two varieties. While corresponding exons of the subfamily B showed 80 to 88% nucleotide sequence homology, those exons were only 60–65% homologous to those of the glutelin A subfamily [15, 19, 24], distinguishing them from the subfamily A. Intron position and derived polypeptide structure, in addition to the nucleotide sequence, confirm the subfamily B members as glutelins. Analysis of RNA from seeds of different stages of development showed that the subfamily B members were expressed at the same time as those of subfamily A, demonstrating coordinated regulation of the two subfamilies.     

Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.     

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.     

The murine 3β-hydroxysteroid dehydrogenase multigene family: Structure, function and tissue-specific expression

The classical form of the enzyme 5-ene-3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3β-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3βHSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3βHSD I and III function as 3β-hydroxysteroid dehydrogenases and 5-en→4-en isomerases using NAD⁺ as a cofactor. The enzymatic function of 3βHSD II has not been completely characterized. Mouse 3βHSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3βHSD I is expressed in the gonads and adrenal glands of the adult mouse. 3βHSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.     

The profilin multigene family of maize: differential expression of three isoforms

Profilin is a small (12–15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays . Two cDNAs isolated from a maize pollen library ( ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA ( ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (>90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30–40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH₂ and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.