The Molecular Evolution of Actin

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.     

Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions

There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions.     

Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys.

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in amino acid changes. Variation in SU was not random but occurred predominantly in five discrete regions. Within these variable regions, a remarkable 98% of the nucleotide substitutions changed the amino acid. These results demonstrate that extensive sequence variability accumulates in vivo after infection with molecularly cloned virus and that selection occurs in vivo for changes in distinct variable regions in env.     

The complete nucleotide sequence of the chick a-actin gene and its evolutionary relationship to the actin gene family

The nucleotide sequence of the chick a-actin gene reveals that the gene is comprised of 7 exons separated by six very short intervening sequences (IVS). The first IVS interrupts the 73 nucleotide 5' untranslated segment between nucleotides 61 and 62. The remaining IVS interrupt the translated region at codons 41/42, 150, 204, 267, and 327/328. The 272 nucleotide 3' untranslated segment is not interrupted by IVS. The amino acid sequence derived from the nucleotide sequence is identical to the published sequence for chick a-actin except for the presence of a met-cys dipeptide at the amino-terminus. The IVS positions in the chick a-actin gene are identical to those of the rat a-actin gene. While there is partial coincidence of the IVS in the a-actin genes with the vertebrate b-actin genes and 2 sea urchin actin genes, there is no coincidence with actin genes from any other source except soybean where one IVS position is shared. This discordance in IVS positions makes the actin gene family unique among the eucaryotic genes analyzed to date.     

Analysis of apolipoprotein E7 (apolipoprotein E-Suita) gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E7 gene from a patient with hyperlipoproteinemia. Apolipoprotein E7 (apolipoprotein E-Suita) is a variant of apolipoprotein E with four additional units of positive charge compared to apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E7/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. Two out of five clones expressed apolipoprotein E7. The analysis of the nucleotide sequence of the exon and exon-intron boundary regions has shown two G-to-A nucleotide substitutions in the 548 and 551 nucleotide positions from the 5'-end of the fourth exon. These two base substitutions change the amino acid residues -Glu-Glu- to -Lys-Lys- at the 244 and 245 positions from the amino-terminus of the mature protein, and give four additional units of positive charge to the molecule.     

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Base substitutions in transposable element IS1 cause DNA duplication of variable length at the target site for plasmid co-integration.

We demonstrate that base substitutions in the IS1 sequence affect the length of the nucleotide sequence which is duplicated during IS1-mediated co-integration. IS1K, an IS1 variant present in the Escherichia coli chromosome, has seven base substitutions in its sequence as compared with that of IS1R derived from the plasmid R100. All substitutions are located in the internal region of IS1K. We have constructed plasmids containing IS1R, IS1K and hybrids between them: one contains four base substitutions causing an amino acid substitution in the insA gene and the other has three substitutions producing an amino acid substitution in the insB gene. We have isolated co-integrate plasmids formed by each IS1 and analysed nucleotide sequences of the target sites duplicated at the co-integration junctions. The results show that IS1K generates duplications of 8 or 14 bp as well as 9 bp, while IS1R exclusively generates the 9-bp duplications. Both hybrid IS1s also create 8- or 7-bp target duplications in addition to 9-bp duplications. These results indicate that the base substitutions in either insA or insB are sufficient for the occurrence of unusual target duplications, suggesting that both genes are involved in the target duplication.     

Phylogeny and substitution rates of angiosperm actin genes

Forty-four actin genes from five angiosperm species were PCR-amplified, cloned, and sequenced. Phylogenetic analysis of 34 of these actins, along with those previously published, indicates that angiosperm actin genes are monophyletic and underwent several duplications during evolution. Orthologues have been identified between Solanaceae species, as well as between Solanaceae species and soybean. These sequences were used to calculate nucleotide substitution rates. The synonymous rate (6.96 x 10(-9) substitutions/site/year) is similar to that of other nuclear protein-coding genes, but the nonsynonymous rate (0.19 x 10(-9) substitutions/site/year) is 6-19 times higher than that of mammalian actin genes. Relative rate tests indicate that actin genes are evolving at similar rates in monocots and in dicots. Evidence is also presented that some members of the maize actin multigene family have been involved in gene conversion events, that the potato genome contains 24 +/- 12 actin genes, and that potato and tomato diverged 11.6 +/- 3.6 MYA.      

Construction of interleukin-1 alpha mutants using unequal contamination of synthetic oligonucleotides.

Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants.     

Nucleotide sequence of the soybean mitochondrial 18S rRNA gene: evidence for a slow rate of divergence in the plant mitochondrial genome

Summary The nucleotide sequence of the 18S rRNA gene from soybean mitochondria was determined and is presented here in comparison to the 18S rRNA genes from wheat and maize mitochondria. All three genes exhibit remarkable sequence similarity supporting the proposal that there is a slower rate of nucleotide divergence in plant mitochondrial DNA (mtDNA) as compared to the mtDNA of animals. A lower degree of sequence similarity is observed between the dicotyledenous plant soybean and either wheat (84%) or maize (85%) than between the two monocots (96%). A possible secondary structure for the soybean 18S rRNA is presented that is analogous to the proposed structure for the E. coli 16S rRNA.     

DNA Sequence Variation within Maize and Melon: Observations from Polymerase Chain Reaction Amplification and Direct Sequencing

While compiling genetic linkage maps in several plant species based upon restriction fragment length polymorphisms (RFLPs), it was noted that the incidence of polymorphism differs among species. The basis of this disparity was investigated in this study by examining the nucleotide sequence at homologous loci among distinct cultivars within two species which exhibit considerably different levels of RFLPs. Using the polymerase chain reaction, homologous regions from different cultivars were first amplified and the nucleotide sequence of the products were determined. Four genomic regions of seven maize cultivars and three genomic regions of eight melon cultivars were examined to compare the respective levels of sequence variation between the two species. Levels of variation for both base substitutions and insertions/deletions varied widely among the maize sequences and between maize and melon for base substitutions. Estimates of theta (a measure of polymorphism) ranged from 0 to 0.002 in melon and from 0.006 to 0.040 for base substitutions and from 0.002 to 0.023 for insertions/deletions in maize. Critical value tests and chi-squared tests suggested that in maize the underlying processes generating and maintaining neutral mutations differ among the regions. The results not only suggest that several mechanisms are necessary to explain the variation seen in these two species, but also point to some basic dissimilarities in the organization and maintenance of the genomes of different plant species.     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

豌豆卷须肌动蛋白Ⅱ类异型体cDNA克隆的序列分析

分析１５个豌豆卷须肌动蛋白ｃＤＮＡ 克隆的限制性内切酶图谱,发现在豌豆卷须中至少存在三类肌动蛋白异型体,分别命名为Ⅰ类（ＰＥＡｃ Ⅰ）、Ⅱ类（ＰＥＡｃ Ⅱ）和Ⅲ类（ＰＥＡｃ Ⅲ）异型体．三类异型体的克隆数目分别为１０、４和１个,表明三类异型体在豌豆卷须中的表达是不同的,很可能具有组织或发育阶段的特异性．对Ⅱ类异型体的三个ｃＤＮＡ 克隆ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１进行了全序列测定,所测定的序列已被ＧｅｎＢａｎｋ 数据库所接受．测序结果表明,ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１的序列长度分别为１５５０、１６８０和１０９１个核苷酸,其中编码区长１１３４个核苷酸,编码的氨基酸长度为３７７（ＰＥＡｃ１１缺少编码氨基端前９６个氨基酸的核苷酸序列）．三个克隆的核苷酸序列完全相同,差别仅在于３′非翻译区的长度不同,即ｐｏｌｙ（Ａ）的加入位点不同．这说明它们可能是由同一基因转录而来,但转录后的加工过程不同．豌豆卷须中肌动蛋白基因ｐｏｌｙ（Ａ）加入位点的使用,可能与组织或发育的特异性表达有关．此外,豌豆卷须肌动蛋白三类异型体之间的核苷酸序列同源性为８０％ ,氨基酸序列同源性为９４％ ．     

Differences in editing of mitochondrial nad3 transcripts from CMS and fertile carrots

A high level of the nucleotide sequence conservation was found for mitochondrial nad3 gene of carrot. Three silent nucleotide substitutions differentiate nad3 open reading frames from cytoplasmic male sterile and male fertile carrots. All these differences are preserved on the RNA level. Partial and silent editing also distinguished both carrots. Three of the C to U conversions were specific to the fertile line. In the two examined carrot lines editing did not affect the mode of alteration of encoded amino acids.     

Actin gene family of Caenorhabditis elegans

Four actin genes have been isolated from Caenorhabditis elegans that account for all of the major actin hybridization to total genomic DNA. Actin genes I, II and III are clustered within a 12 X 10(3) base region; gene IV is unlinked to the others. All four genes have been sequenced from at least nucleotide -109 to +250. Genes I and III are identical for the first 307 coding nucleotides. Genes I and II differ in 14 positions within the first 250 coding nucleotides; one difference substitutes an aspartic acid for a glutamic acid at codon 5. Genes I and IV differ in 18 positions within the first 259 coding nucleotides without causing any amino acid differences. Genes I, II and III have introns after the first nucleotide of codon 64 and gene IV has an intron between codons 19 and 20. The four nucleotide sequences thus far define two different amino acid sequences. Both of the amino acid sequences resemble vertebrate cytoplasmic actin more than vertebrate muscle actin. A DNA polymorphism between the Bristol and Bergerac strains has been used as a phenotypic marker in genetic crosses to map the cluster of actin genes within a 2% recombination interval on linkage group V between unc-23 and sma-1 in order to begin a molecular genetic analysis of the actin loci.