Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: I. Genetic mapping and DNA sequence

A new bacteriophage T4 gene has been identified and located between genes 20 and 21. This gene codes for PIP, a component of the prehead core and precursor to one of the two species of small, acidic peptides found inside the mature phage head. We have determined the DNA sequence of the gene. Both the DNA sequence and the amino acid sequence derived from it are unusual, and between them explain why suppressor-sensitive mutations in the gene have not been found using classical mutagenesis. The codon usage in this gene is highly non-random. In the accompanying paper we show that PIP is essential for T4 growth and assign its gene a number. 67, to indicate that fact.     

A conserved nucleotide sequence, coding for a segment of the C-propeptide, is found at the same location in different collagen genes.

The nucleotide sequence of a segment of the chick alpha 1 type III collagen gene which codes for the C-propeptide was determined and compared with the corresponding sequence in the alpha 1 type I and alpha 2 type I collagen genes. As in the alpha 2 type I gene the coding information for the C-propeptide of the type III collagen gene is subdivided in four exons. Similarly, the amino proximal exon contains sequences for both the carboxy terminal end of the alpha-helical segment of collagen and for the beginning of the C-propeptide in both genes. Therefore, this organization of exons must have been established before these two collagen genes arose by duplication of a common ancestor. In several subsegments the deduced amino acid sequence for the C-propeptide of type III collagen shows a strong homology with the corresponding amino acid sequence in alpha 1 and alpha 2 type I. For one of these homologous amino acid sequences, however, the nucleotide sequence is much better conserved than for the others. It is possible that a mechanism of gene conversion has maintained the homogeneity of this nucleotide sequence among the interstitial collagen genes. Alternatively, the conserved nucleotide sequence may represent a regulatory signal which could function either in the DNA or in the RNA.     

Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene

Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein. The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions. Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported. In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx. 3.3 kb downstream from AGP1. The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences. It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

New mre genes mreC and mreD, responsible for formation of the rod shape of Escherichia coli cells.

New shape-determining genes in the mre cluster at 71 min on the Escherichia coli chromosome map, named mreC and mreD, were identified by complementation experiments using delta mre-678 mutant cells, which have a 5-kilobase-pair deletion encompassing the mre region, and by DNA sequencing. The delta mre-678 mutant cells required three genes, the previously reported mreB gene and the two new genes, to restore the normal rod shape of the cells and normal sensitivity of growth to mecillinam. The mreC gene is preceded by the mreB gene and by a 65-base-pair spacing sequence containing a palindrome sequence and a possible Shine-Dalgarno sequence. The deduced amino acid sequence of the MreC protein consists of 367 amino acid residues with a molecular weight of 39,530. The initiation codon of the mreD gene overlaps the termination codon of the mreC gene by one nucleotide residue. The deduced amino acid sequence of the MreD protein consists of 162 amino acid residues with a molecular weight of 18,755. In vitro, the coding frames of mreC and mreD produced proteins with Mrs of 40,000 and 15,000, respectively, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases.

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

The amino-acid sequence of a purothionin from Triticum monococcum, a diploid wheat

Purothionins were extracted and purified from the diploid wheat Triticum monococcum. Two proteins were obtained, one of which was present in only very small amounts. The major purothionin of T. monococcum was sequenced and it had an amino acid sequence identical with that of the beta-purothionin of Triticum aestivum (hexaploid bread wheat). It is known that T. monococcum contains the wheat A genome, so the structural gene coding for the beta-purothionin must comprise a part of the A genome. There have been no observable (as amino acid replacements) changes in the DNA comprising either the beta-purothionin gene of T. aestivum or the purothionin gene of T. monococcum, since T. monococcum (or its wild equivalent, Triticum boeoticum) hybridized with the diploid wheat B genome progenitor and started the evolution from diploid to allohexaploid wheat. All of the investigated characteristics of the purothionin-like protein isolated in small amounts suggested that it was essentially identical in amino acid sequence with the T. monococcum purothionin. It may be a dimerized form of beta-purothionin.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

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.     

The organization of the prosystemin gene

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.     

Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence. Homology to Na+/K+-, Ca2+-, and K+-ATPase

The gene for the Neurospora crassa plasma membrane H+-ATPase has been cloned and sequenced. The gene encodes for a protein of 920 amino acids with a molecular weight of 100,002. The coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus. The deduced amino acid sequence of the N. crassa plasma membrane H+-ATPase exhibits 75% homology to the amino acid sequence of the Saccharomyces cerevisiae plasma membrane H+-ATPase. Also, an amino acid comparison with the Na+/K+-ATPase from sheep kidney, Ca2+-ATPase from rabbit muscle, and K+-ATPase from Escherichia coli reveals that certain regions are highly conserved and suggest that these regions may serve essential functions which are common to the various cation-motive ATPases. This observation suggests that the phosphorylatable, cation-motive ATPases may function via a similar energy transduction mechanism.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。