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Determining the sequence of parasite DNA
T@Gu”in!qu@B Determining the Sequence of Parasite DNA G.R. Reddy Know/edge ofthe biochemical mechanisms critical for the compiet,on ofo parasite’s I+ cycle is fundamental to deveioplng controi strategies. However, most of the studies of parasiteproteins are limIted by the d$ ficulty in obtaining suficient amounts of purified proteins for b/ochemica/ anolys~s. Alternatively, parasite genes can be cloned and sequenced, then the sequence information used to prepare large qua&es of recombinant proteins fir b/ochemica/ analysis. Roman Reddy brief/y summarizes some ofthe commonly employed methods to isolate parasite genes, based on protein function and expressjon pattern of the genes. He then eiaborates on the random sequence approach, with special refirence to Plasmodium falciparum. Recent advances in molecular biology have revolutionized the rate at which sequence information is obtained from parasites. Typically, sequence data are generated from a clone isolated by screening a complementary DNA or genomic library constructed in E. co/r using a plasmid, bacteriophage or cosmid vectors. Although it is possible to prepare such libraries for most parasites, very few reports describe libraries with large segments of Piasmodium folciparum DNA. This is because of the extensive rearrangements of the DNA in E. co/i’, which may be caused by the parasite’s unusually high A+T-rich genome (A+T content is -82% for the entire DNA, and -90% for non-coding regionsiJ). The preparation of stable genomic libraries for P. faiciparum in E. CO/I is possible3,4 using the genease activity of mung-bean nuclease, discovered a decade ago3. Under modified reaction conditions, mung-bean nuclease cleaves P. falciparum genomic DNA and releases gene fragments with complete or partial coding sequence+5. However, libraries prepared ustng this strategy do not represent the entire genome, since noncoding sequences are greatly reduced in the DNA treated with mung bean nuclease. Large fragments of P. falciparum DNA can also be packaged in yeast using the yeast artificial chromosome (YAC) vector+7. In addition to cloning genes, sequence data are obtained by PCR (polymerase chain reaction) ampllficatlon of a gene using a set of oligonucleotide pnmers forasitology Today, vol. I I, no. I, i 995
prepared from conserved regions of the gene*. This is frequently performed for ribosomal RNA genes to accomplish phylogenetic analysis. Generally, parasite genes are cloned based either on their function or the expression pattern of the proteins they encode. However, not all genes can be cloned on the basis of functional properties, since the functions of many genes are unknown. Alternatively, sequencing genes at random allows sequence determination irrespective of their function. The following are some of the commonly employed strategies to clone and sequence parasite genes. Cloning Methods Based on Protein Function Nucieic acid probes. Proteins with similar functions are known to have several conserved regions in their primary structure, which is the basis for isolating genes using heterologous nucleic acids as hybridizing probes. Because of their expected degree of homology, hybridizations with heterologous probes are usually performed under low-stringency hybridization and washing conditions. However, such conditions often result in the cloning of several undesired genes. This method has other constraints: (I) It cannot be used to clone genes unique for a parasite since it is impossible to obtain a probe; (2) a gene of interest must be available from another organism, preferably from a closely related species, to increase the probability of Isolating the gene before initiating the study, and (3) an additional limitation to cloning P. faioparum genes is Its unusual codon usage derived from the parasite’s high A+T content’? As a result, only SIX P. faicrparum genes have been cloned using heterologous probes (see Table I ), Degenerate oiigonucieotldes. Alternatively, protein sequence data can be used to synthesize degenerate oligonucleotides as hybridizing probes. Typically, oligonucleotides are synthesized by back-translating an established protein sequence encoded by the desired gene. However, the protein sequencing depends on the availability of pure protein. Purifying proteins from P. fobparum or other intracellular parasites IS @I995
E swer Sc~enie Ltd 0 I 69-475819 j/$09 50
a very time-consuming and tedious process. To date, aspartyl hemoglobinase is the only protein purified from P. /doparum using conventional purification methods9, which require the propagation of parasites in very large quantities. Subsequently, the sequence data obtained from the purified protein are used to clone the genelo. If sequence information is available from several species for a gene, oligonucleotides can be designed from the conserved regions of the gene. The conserved regions are typically identified by comparing all the sequences. Such oligonucleotides are used as hybridizing probes or PCR primers to clone the gene from another species. The majority of P. folciparum genes are cloned by these methods (Table I). The length and degeneracy of the oligonucleotides play an Important role. Hyde et al.’ I suggest that the optimal length of an oligonucleotide for isolating P. falciparum genes is 35-42 bases. When selecting oligonucleotides, it IS ideal to include amino acids encoded by few codons, in order to minimize the degeneracy. Particularly for PCR primers, the ideal amino acid at the 3’ end of a primer is Met or Trp (each having only one codon), thereby increasing the probability of amplifying the target gene. PCR can also be employed using one degenerate oligonucleotide to the gene of interest to obtain sequence information. In this approach, a library of DNA fragments ligated into a vector are amplified using a degenerate ollgonucleotide to the gene and an oligonucleotide to the vector sequence flanking the gene as PCR pnmers12. Such a method is vet-y valuable for genes in which only one conserved region is identified. However, the major problem in using degenerate oligonucleotides is their false priming to undesired sequences, resulting in the cloning of false positives. False positives can be minimized by performing a second PCR reaction on the fint PCR product using nested primers. Nested primers can be designed from another conserved region internal to the amplified product to increase the specificity of the PCR. functiona/ complementation. Functional properties of gene products are also exploited to isolate genes by genetic 37
Techniques complementation. A functional recombinant protein of a parasite gene can complement a host that lacks the respective functional protein. The genes for citrate synthetase gene of Coxiella burner;; 13, phosphoglycerate kinase of Trypanosoma bruceii4, phosphofructokinase and phosphoenolpyruvate carboxykinase of Hoemonchus contortus Is,’$ triosephosphate isomerase of Giardio /amb/,a17 and glucose phosphate isomerase and ornithine aminotransferase of P. falciporum’* have each been cloned by complementation of E. co/imutants. The adenine phosphoribosyltransferase gene from P. fakiporum has been cloned in mutant mouse L cells that lack the enzyme’9. The major advantage tn this approach is the greater success of cloning a complete, functional gene. However, availability of a suitable mutant in a host is the primary step that often is the major limitation. Antisera. Antisera raised against a protein can be used to isolate the gene encoding that protein from a library constructed in an expresston vector. This approach has two limitattons: (I) large quantities of purified protein are required to raise antisera; and (2) antisera raised against a protein often crossreact with nonspecific antigens. To date, the only metabolic enzyme gene cloned from P. fdciparum by this method is lactate dehydrogenase? Since the major research emphasis on malaria is to develop a vaccine, most of the genes sequenced from P. falciparum encode immunodominant surface antigens. Immune sera from individuals repeatedly infected with malana parasites are used to clone antigen genes, and the majority of antigen genes of P. filciporum are cloned by this method. Nearly 90 different antigen genes/gene fragments have been sequenced, and are listed in the UNDP/World Bank/ WHO /TDR program malaria database available from the World Health Organization. Most of them encode antigens that contain several amino acid repeats. Because of the variation in the repeats2’ of proteins from parasites collected from different locations, such antigens are less likely to be vaccine targets. Thus, the development of alternative methods to clone nonrepetitive antigen genes to which the host develops consistent immunological responses is essential. Cloning Expression
Methods Pattern
I. Plosmodium ~~/C$WWTJ structural
conventional
genes with
known
function
cloned
by
methods
Gene Heterologous Actin
nucleic acid probes
Accession no. a
Year
Ml9146
I988
Calcium ATPase
X7 I 765
I993
Calmodulin
M59770
1991
YOO5I9
I987
P-tubulinb
M3 I205
I989
y-tubulin
X62393
I993
X65738
I993
X61921
1991
JO3028
I987
Hypoxanthine-guanine
phosphoribosyl transferase
Degenerate oligonucleotide Cation ATPase CDC2
probes
protein kinase
Dihydrofolate
reductase-thymidylate
synthetase
DNA
polymerase (Y
L I8785
1991
DNA
polymerase ab
X62423
1991
M24322
I989
X63648
I992
RNA polymerase I
LI II72
I993
RNA polymerase II
Xl6561
I989
RNA polymerase Ill
M37820
1991
Topoisomerase
x79345
I’994
cw-tubulin I
x I 5979
I 989
c-Y-tubulinII
M34390
I 990
P-tubulinb
X I 6075
I 989
Unusual protein kinase
X67288
I 993
Multidrug resistance gene
I (PfMDR I )b
Protein kinase
II
Degenerate oligonucleotide Aspartic hemoglobinase
PCR primers X75787
I 994
Cysteine proteinase
M80590
I992
Dihydroorotate
JO3028
I987
M647 I 5
1991
ERD2
X74869
I993
Hexokinase
M92054
1992
Multidrug resistance gene I (PfMDRl)b
M24850
I989
Multidrug resistance gene 2 (PfMDR2)
M2485 I
1989
3-Phosphoglycerate
DNA
dehydrogenase
polymerase sb
M59249
1991
reductase I
L22057
I993
Ribonucleotide reductase 2
L22058
I993
Triosephosphate
LO 1654.5
I993
LO8200
I993
JO5544
I990
L I 5426
I993
JO3084
I988
M IO985
I985
M I9753
I986
JO4072,3
I988
Heat shock protein
X I5292
I989
Lactate dehydrogenase
Ml4818
I985
X60488
1991
M80655
1991
Xl3014
I988
Ribonucleotide
kinase
isomerase
Vacuolar ATPase Functional complementation Glucose phosphate isomerase Ornithine
aminotransferase
Antisera Aldolase Glycophorin-binding
protein
Heat shock protein 70 (HSP70) Heat shock protein 78 (glucose-regulated
protein)
No methods are described for these genes Elongation factor I (Y Glucose-6-phosphate
dehydrogenase
Heat shock protein 90 (HSP90) a Accession no. and year represent the initial discovery of the gene. b Gene isolated by more than one method.
Based on the of the Genes
Differential screening and subtractive hybridization techniques. Genes expressed in different life cycle stages of a 38
Table
parasite can be cloned by using the differential hybridization screening technique. In this method, labelled cDNA prepared from mRNA of different developmental stages are hybridized sequentially to
a library of the parasite. Clones unique to each stage can then be identified by their differential hybridization patterns. The major advantage of this approach is that it does not requtre prior knowledge Parasitology Today, voi. / I, no. I, I 995
Techniques of the genes. For example, many developmentally regulated genes have been cloned from promastigote/ amastigote stages of Leishmon,o majorZ2 and L. donovc~ni23,and also from Eimetio bovis24.
This method is also appropriate for cloning strain-specific genes. For example, Entomoeba h~stolytica causes amebic dysentery in only 10% of the people infected with this parasite. This is due to variations in the virulence of different parasite strains. By using this method, a strain-specific gene for this parasite has been cloned25. However, this method is only applicable for highly expressed stage- or strain-specific genes. To clone genes expressed at low levels, one needs to utilize additional methods such as the subtractive hybridization technique. In this method, mRNA from one developmental stage is hybridized with cDNA from another stage of the parasite, and the unhybtidized mRNA is separated and used to screen a library. For example, a cyclic AMP-inducible gene from the infective stage of Tryponosoma cruzl is isolated in this mannerz6. Nevertheless, these methods are not very effective for pre-erythrocytic stage genes of P. folciporum, since it is dificutt to isolate mRNA from mosquito or liver-stage parasites that is free from host RNA. Subtractive hybridization technique is also used to clone DNA unique for a genome which is valuable to determine DNA polymorphisms within a parasite species. Representative Difference Anaiysis. Recently, Lisitsyn et a/.17 developed a system, called representative difference analysis, to clone the differences between two complex genomes. In this method, subtractive and kinetic enrichment is used to purify restriction endonuclease-digested fragments unique for a genome. Genomic DNA from two sources with genetic variations are digested with a restriction endonuclease. Then, oligonucleotide adaptors are ligated to one genomic DNA (tester), and annealed to an excess of the other (driver). Subsequently, the DNA is amplified by PCR using oligonucleotlde primers to adaptor sequence. Under these circumstances, DNA fragments unique for the tester DNA are the only ones that contain adaptor sequence on both ends, which allows the exponential amplification of the unique DNA. DNA conserved in both the genomes can only be amplified in a linear fashion, since only one of the two strands can have adaptor sequence on them. Consequently, the exponentially amplified PorositologyToday, vol. I i, no. I, I 995
DNA IS cloned. Such a method may be useful for the rapid identification of regions of DNA linked to genetic rearrangements in parasites. Differential display. Recently, Liang and Pardee2a have developed another method, called differential display, tnvolving PCR that rapidly identifies differentially expressed, low-abundance genes of higher eukaryotes. In this method, mRNA is amplified by using a set of oligonucleotide primers, one primer being annealed to the polyadenylate tail of a subset of mRNA, and the other being short (ten bases) and arbitrary in sequence, so that it anneals at different positions relative to the first primer.. PCR products from different sources are then compared on a DNA-sequencing gel to identify genes expressed differentially. Although not used for parasites, this method may be effective to identify some stage-specific genes. Random Sequencing Approach Expressed sequence tags (ESTs). Recent reports on random sequencing of cDNA libraries from human brainI and Caenorhabditis elegans30 have shown that it is an efficient method for obtaining preliminary data on coding sequences In this method, automated DNA sequencers and computer programs are used to obtain large amounts of sequence information, Recently, we have reported 389 ESTs (GenBank accession numbers T02472-TO2633 and T 17984-T 18255) derived from 550 cDNA clones of the etythrocytic stage of P fabparum3’. Database searches of these sequence tags to nucleic acid and protein sequence databases have led to the putative identification of 90 unique genes (Table 2), of which 28 are similar to previously reported P. folciparum genes. By analyzing the types of genes identified, it is evident that this approach is effective for detecting highly expressed housekeeplng and stagespecific transcripts (Table 2). However, additlonal steps to reduce the clones derived from abundant messages In the library are needed. Such steps will increase the probability of identifying lowcopy, regulatory and stage-specific genes. Another problem with the random cDNA approach is the high redundancy rate (28% In the case of P. f&parum3~). However, this redundancy can be minimlzed by Including addittonal screentng steps to exclude clones for whtch sequence tags are available. Since pure mRNA is not readily available from preerythrocytlc stage parasites, it is not
possible to prepare ESTs from these stages. Gene sequence tags {GSTs). Alternatively, random sequencing of genomic DNA may allow the identification of genes from all life cycle stages. But given that only 60% of the genome of Plasmedium is unique32, the identification of ‘real’ genes is complicated by the presence of non-coding sequence. Recently, we have published another report that describes the strategy by which one can efficiently generate sequence data for P. falciparum genes that overcome some of the problems associated with random sequencing of cDNA and genomic DNA4. In this report, we described the strategy to sequence genomic DNA in order to generate GSTs efficiently. Plasmodium falciparum genomic DNA was digested with mungbean nuclease, which IS used to prepare a plasmid library in E. co/i (mung-bean nuclease cleaves P. fa/c$afum genomic DNA and releases gene fragments with complete or partial coding sequence&s). A large number of recombinant clones were selected at random from this library and sequenced to generate sequence tag data. A total of 673 unique sequence tags were obtained from 400 clones (average length of each tag is -320 nucleotides). These data were reported in the GenBank (accession numbers T02634-TO2808 and TO9496-T09993). The database search of these GSTs revealed the putative identification of 5 I unique genes (Table 3) of which only five encode prev~ously reported P. falciparum genes. The genes identified In this study include proteins expressed in etythrocytic, exoerythrocflic and sexual stages of the parasite. This approach clearly allows the isolation of genes expressed in all stages of the parasite. The PREDICT computer program, based on patterns of codon usage and amino acid composition of P. falciparum genes, identifies open reading frames (ORFs) specific for P. fa/ciparum33. Analysis of GSTs by this program indicated that nearly 90% of the clones with no putative identification have long ORFs. Therefore, the majority of the clones sequenced are likely to be new genes with unknown function. In conclusion, this method offers the promise of obtaining partial sequence data for all the parasite genes at a rapid rate. Although this method is tested only for the P. falciparum genome, it is possible to determine large-scale sequence data for genomes of other parasites by this approach. Genes from many other protozoans, including Trypanosoma34, 39
Techniques Table 2. Plosmodium fakiparum
expressed
sequence tags having significant homology Clone
with database sequences
I
Clone name 01 l4C
Accession no. TO2509
Putative identification 60s acidic ribosomal protein P2
47
0807C
;;8197
Laminin receptor
2
0079c
TO2490
AAC-rich
48
023OC
TO2586
Late-stage histone H3, H4
3
0286C
TO2627
Actin la
49
OIOOC
TO2497
Lipoic acid synthetase
4
0285C
TO2626
Activator
50
0325C
T I8003
Major merozoite
5
Ol47C
TO2534
ADP-ribosylation
51
0682C
Tl8127
Mature-parasite
6
Ol54C
TO2540
Alanine tRNA
52
0412c
T I8048
7
036OC
Tl8018
Aldolase’
Membrane associated Ca-binding protein+
8
016lC
-
Asparagine-rich protein”
53
0676C
Tl8122
Merozoite-specific
9
0283C
TO2625
ATP-dependent
54
0289C
TO2628
Methionyl tRNA
No.
protein I 37 kDa subunit factor 4
synthetase
RNA helicase
No.
name
Accession
.
Putative
identification
surface Ag” infecteda
surface Ag” synthetase
IO
0309c
T I 7990
Breast basic conserved protein
55
Ol3lC
TO252 I
Modifier
II
025lC
TO2605
Calcium-binding protein”
56
0075c
TO2486
Multicatalytic endopeptidase Nucleolar transcription factor
12
0322C
Tl8001
Calmodulin”
57
03ooc
T I 7986
13
01 I IC
TO2506
Casein kinase II alpha
58
OOOIC
TO2472
Nucleolin
14
0706C
Tl8140
CCAAT-binding
59
0327C
T I 8004
ORF
factor
I protein
15
038lC
T I8027
cdc2 I protein
60
0464C
T I8079
Phenylalanyl tRNA
16
0856C
T I8228
Cell division control protein 48
61
08OOC
Tl8193
Pr86 rhoptry-associated protein I”
I7
OlO3C
TO2500
Choline kinase
62
0333c
T I8008
Prohibitin
I8
038OC
T I8026
Circumsporozoite-related
63
0648C
Tl8107
Protein kinase A catalytic chain
I9
0228C
TO2585
Deoxyribonuclease
64
0446C
T I8067
Protein kinase C inhibitor
20
Ol63C
TO2546
Elongation factor I B
65
0757c
Tl8165
RAP-2a
21
OlO5C
T02501,2
Elongation factor 2
66
0224C
TO258 I
RD protein
22
0496C
T18096
Embryo-specific dormancy protein
67
0663C
Tl8l
Rhoptry-associated
23
0787C
Tl8181
Erythrocyte-binding
68
0367C
T I8022
Ribonucleoside-diP reductase
24
0737c
Tl8156
Gene I I-la
25
0792C
Tl8185
Gene fiu
69
0293C
TO2809
(large) Ribonucleoside-diP reductase (small) Ribosomal protein L9
antigen”
proteina
I protein
I5
synthetase
protein
Ia
26
0812C
T I8202
Genes rp0I-f. rpoS
27
05ooc
T I8099
Glucose-6-phosphate
70
Ol95C
TO2564
28
0678C
Tl8124
Glutamic acid-rich protein”
71
049lC
T I8094
Ribosomal protein S9
29
007lC
TO2482
Glyceraldehyde-3-P-dehydrogenase
72
0312C
T I 7993
RNA helicase
30
0675C
T18253
Glycerol-3-P-dehydrogenase
73
0799c
Tl8192
S-adenosyl methionine synthetase
74
0892C
isomerasea
S-antigen precursora
31
Ol65C
TO2548
Glycine tRNA
32
01 l3C
TO2508
Glycogen synthase kinase-3
75
0337c
Tl8012
Sec23
33
0252C
TO2606
Glycophorin-binding
76
0766C
Tl8168
Serine-rich proteina
34
0237C
TO2592
GSTI -HS GTP-binding protein
77
04lOC
T I8047
Serine-rich protein
78
OlO8C
TO2504
Serine-rich protein homologue”
synthetase proteina
35
0709c
Tl8143
Heat shock protein 82
36
Ol57C
TO2543
HGPRT”
79
048lC
T I8087
Splicing factor SC35
37
0418C
T I8054
Histone H2B
80
065OC
Tl8108
Suppressor 2 of zeste
38
076lC
-
Histone H3
81
008OC
TO249 I
T-protein
007oc
TO248 I
TAT-binding
protein
Thioredoxin
precursor
39
0212c
TO2575
HSP70”
82
40
036lC
Tl8019
HSP90a
83
034lC
Tl8015 T18023
Threonyl tRNA synthetase
41
0072C
TO2483
HSF90-B (HSP84)
84
0369C
42
0364C
-
Interspersed repeata
85
Ol85C
86
0789C
Tl8182
Transketolase
87
0795c
Tl8188
Troponin C
88
OIOIC
TO2498
Ubiquitine-conjugating
89
Ol28C
TO25 I9
Vacuolar
ATPase
90
069OC
Tl8130
Vacuolar
ATPase”
43
0323C
T18002
lsoleucyl tRNA
44
0233C
TO2588
Keratin
45
Ol86C
-
Knob-associated protein”
46
0223C
TO2580
histidine-rich
Lactate dehydrogenase”
“Tags having homology to previously reported
protein
enzyme
f. fokiparum genes.
Giardio35, Toxogkwno36, Leishmanio37 and Babesia38,39, have been cloned by using the genease activity of the mungbean nuclease. These reports suggest that such an approach is feasible for these parasites. The major limitation with randomsequencing methods is that It WIII take a long time to identify a specific gene. The probability of isolating a specific gene at random from a P. falciparum gene fragment library is less than one In 40
synthetase
Transformation-sensitive
7500 clones (assuming there are 7500 genes, and all of them are represented only once). However, analysis of our data4 indicates that only one in three clones analyzed have complete coding sequence for genes; the remaining two-thirds have one or more exons. In addltlon, IO% of the clones are redundant clones. These variables reduce the chance of Isolating a gene to less than one out of I4 000 clones sequenced.
Conclusion Approximately 40 metabolic enzyme genes and 90 surface antigen genes of P. folciparum have been isolated by conventional cloning methods. The data obtained by random sequencing methods represent over 800 new genes (of which 108 have significant similarity to genes reported in databases). Most of the data from random sequencing have been obtained, in less than two years’ Parasitology
Today, vol.
I I, no. i, I995
Techniques Table 3. Plasmodium folciparum gene sequence tags having significant homology with database sequences No. I
2 3 4 5 6 7 8 9 IO II I2 13 I4 I5 I6 17 18 I9 20 21 22
TO9726
Putative identification 305 ribosomal protein S- I8 30s ribosomal protein S I2 50s ribosomal protein L I6
TO962 I ,2
605 ribosomal
protein
Ll8A
TO993 I
60s ribosomal
protein
L27a
T09807,8
60s ribosomal
protein
L8
T02745,6
Actin
T09692,3
Adenylyl
TO96 I7,8
Alternative
T09594,5
ATP-dependent
T09922.3
/3 coat protein
T09635.6
Bkm-like sex-determining region hypothetical protein CS3 I9
Clone name 0026M 0368M 0324M 0204M 0482M 0390M 0088M 0284M 0202M 0187M 0470M 021 IM
Accession no. T02656,7 TO978 I ,2
0487M 0075M 0405M 0017M 0362M 0291M 0422M 0431M 0132M 0145M 0136M 0419M 0065M 0421M 0058M 0197M 0225M
T09936.7
Ca’+-dependent
T02726,7
Cathepsin
D (aspartyl proteinase)
T09822,3
Clustered
asparagine-rich
TO2647
Cyclophilin
TO9772
DNAJ protein
T09700, I
DNAJ
T09844,5
DNAJ protein
T09860, I
Duplicate
T02798,9
Dynamin
Ila cyclase gene splicing factor- I RNA helicase
protein
kinase” protein
(Bacillus subtilis) (& cob)
protein
homologue
HSJ I
procyclin
T09530, I
Dynein P-chain
T02796,7
Glucose-6-P
TO984 I
Glutamate
TO271 I.2
Glycerol-3-P
dehydrogenase
T09842.3
GTP-binding
protein
RYH I
TO2702
GTP-binding
protein
YPT3
T09608,9
Initiation
T09654,6
Iron-responsive protein
T09847,8
Lactate dehydrogenase
T09652,3
Liver-stage
32
0424M 0224M 0522M
TO996 I
Mitochondrial protein
33
0175M
TO9580
Mitochondrial pyridine nucleotide transhydrogenase p subunit
34
0314M 0488M 003lM 0158M 0160M
T09709, IO
Nitrogen
T09938.9
PolyA nuclease
T02666,7
Proteasome
TO955 I ,2
Ras-like protein
T09555,6
Regulatory protein cerevisioe
23 24 25 26 27 28 29 30 31
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0188M 0229M 0347M 0337M 0176M 0046M 0201M 0104M 0334M 0167M 0343M 0049M 0543M
“Tags having homology
isomerasea dehydrogenase
factor elF-4A element-binding
antigen (LSA- I)” phosphate-carrier
fixation-U
protein
C3 (Rat) TC4 - Sacchoromyces
T09596,7
Rfbf protein
TO9657
Ribonucleotide
T09752,3
Ring-infected
T09736,7
5. cerevisiae gene (vasa protein)
- 5. typhimurium reductase surface antigen
TO958 I ,2
5. pombe cdc2 I gene
T02688,9
Serine hydroxy
TO96 I5,6
Serine proteinase
T02767,8
Sexual-stage
methyl transferase type
I precursor
specific protein”
T09733,4
Sto~hylococcus xylosus BBM3XM
T09568,9
Thioredoxin
T09744,5
Thrombospondin
T02692.3
Tubulin
T09987,8
Ubiquitin-carrier
to previously
reported
time, by a small group of investigators. Thus, the random sequencing approach provides sufficient sequence information Parasitology Today, voi. i I, no. I, I995
P. fokiparum
precursor
II (c~) protein
genes.
for each gene at a rate that is useful to initiate a detailed study. Numerous new avenues of research into the bio-
chemistry of the parasite have been opened using the genes identified to date by random sequencing approach, which may lead to targeted drug development. The sequence tags with no putative identification are also valuable resources for initiating studies to Identify new antigen genes. The sequence data from the random sequencing project are recorded in the Genbank database and the clones are readily available to initiate such studies. In addition, these tags can serve as a valuable tool in recent efforts40 to construct fine resolution maps of P. folciparum chromosomes. In summary, a gene of interest can be cloned by one or more methods as described in this review. Isolation of a specific gene is easier by conventional methods compared to the random sequencing approach. However, the random sequencing approach provides the opportunity to obtain sequence information of all the genes of a parasite such as P. faiciparum, in three to five years. This should lead to the development of large-scale basic and applied research programs aimed at developing strategies for both chemotherapeutic and immunological control. Acknowledgements I wish to thank Ky Minh
Tran and Suhasin Ganta for thelr assistance In preparing this manuscript. My thanks are also extended to Ray Kaplan, Debopam Chakrabartl, John B. Dame and David R. Allred for their helpful discussions. This work was supported by the Univenity of Flonda, Gainesville, USA.
References
I Weber, J.L. ( 1988) Exp. Porosrtoi. 66, I43 I70 2 Pollack, Y. et a/. (I 982) Nucierc Acids Res IO, 539-546 3 McCutchan, T.F. et al. (I 984) Scrence 225, 625-628 4 Reddy, G.R. et al. (I 993) Proc. Notl Acad Scr USA 90,9867+987 I 5 Vemtck, K.D. et ai (I 988) Nucleic Acids Res. 16, 6883-6896 6 T&x, T. and Kemp, D.J. (I 99 I) Mol. Blochem. Porawtol. 44, 207-2 I2 7 de Bran, D. et 01 (I 992) Genomlcs 14, 332-339 8 Wllks, A.F.( 1989) Proc Not1 Acod So USA 86, I603-l 607 9 Goldberg, D.E. et oi. (199l)j. Exp. Med. 173, 96 l-969 IO Francis, S.E. et 01. (I 994) EMMBOJ. 13, 306-3 I7 I I Hyde, J.E. et al. (I 989) Mol. Biochem. Parosrtoi. 32, 247-262 I2 Oberle, 5.M and Barbet, A.F. (I 993) Gene I36, 29 I-294 13 Heinren, R.A. and Mallavla, L.P. ( 1987) In@ct. Immun. 55, 848-855 I4 Alexander, K. et 0). ( 1990) Gene 90, 2 15-220 R.D. et 41. (199 I) Mol. Blochem. I5 Klein, Parasrtol. 48, 17-26 R.D. et 01. (1992) MO\ Blochem. I6 Klein, Porasrtoi 50, 285-294 17 Mowatt, M.R. et aI (1994) txp Porosltoi 78, 85-92 41
Techniques (I 990) J. 5101.Chem. 265, 12337-12341 Pollack Y. et al. (I 985) Exp. Parasftoi. 60. 270-275 Simmons, D.L. et 01. (I 985) Mol. Blochem. Poras,tol. 15, 23 I-243 Kemp, D.J. et 01. (I 990) Adv. Parasrtol. 29, 75-149 Coulson, RM.R. and Smith, D.F. (I 990) Mol. 6,ochem. Parasrtoi 40, 63 -76 Joshl, M. et al. (1993) Mol. Bfochem. Parasltoi. 58,345-354 Abrahamsen, MS. et al. (I 993) Moi Blochem. Parasitol. 57, I ~ I4 Burch, D.J. et al. (I 99 I) 1, C/in. Mlcrobiol. 29, 696-70 I Heath, S. et al. (I 990) Moi Biochem. Parosrtoi. 43, 133-142
I8 Kaslow, D.C. and Hill, S. 19 20 21 22 23 24 25 26
Plant Parasitic Nematodes in Temperate Agriculture edited by
K. Evans, D.L. Trudgill and
JM. Webster, CAB International, 1993. f75.00 {xi + 648 pages) ISBN 0 85 I98
808 3
Plant nematology has changed dramatically during the ten yean since the last comprehensive book cataloging plantnematode interactions was published’. Many chemical-control practices for nematode pests have either been eliminated or severely curtailed in thetr application due to increased environmental and health awareness. In addition, intensive agricultural production systems have exacerbated existing nematode problems and resulted in the identification of new interactions. As crop damage due to plant parasitic nematodes has increased, funding for research and development of new control practices has dwindled. In spite of this last problem, the past decade has been a period of tremendous excitement and promise in several areas of plant nematode research. It is quite appropriate that these advances be incorporated into a generally available and accessible form. This book provides a comprehensive look at plant-parasitic nematodes attacking a wide range of crops grown in temperate climates, and is a companion volume to the previously released treatise on tropical agriculture2. As such, these two volumes provide an overview of the current status of plant parasitic nematode damage and control in world agricultural systems. The I7 chapters in this book cover both major and minor crops grown in temperate climates, including omamentals, bulb crops, glasshouse crops, mushrooms, and forest trees. Most of the listed commodities 42
27 Lisitsyn, N., Lisitsyn, N. and Wigler, M. (I 993) Soence 259, 946-95 I 28 Liang, P. and Pardee. A.B. (I 992) Science 257, 967-97 I 29 Adams, M.D. et al. (1992) Nature 335, 632-634 30 Waterston, R. et al. (I 992) Nature Genet. I, 114-123 31 Chakrabatii, D. et a/. (1994) Mol. Blochem. Parasitol. I 66, 97-l 04 32 Dore, E. et al. (I 980) Mol. Biochem. Parasitol. I, 199-208 D. (1988) Mol. 33 Saul, A. and Battistutta, Blochem. Parasitol. 27, 35-42 34 Brown, K.H. et al. (1986) J. Bioi. Chem. 261, 10352-10358 35 Adam, R.D. et al. (I 989) 1. Exp. Med. 167, 109-l I8
have been overlooked in previous books in favor of the more traditional agricultural crops, but given the changing nature of production in the 1990s it is appropriate that they are Included here. Of course, the major row and vegetable crops are also covered in this book In addition to the chapters on nematodecrop interaction, there are treatments on nematode extraction and identification, nematode population dynamics and yield loss, molecular diagnostics, quarantine, molecular approaches to control, and insect parasitic nematodes. These are all well-written and useful treatments (particularly the extraction and population dynamics chapters), but the lone chapter on insect nematodes seems a bit out of place in this particular venue. One chapter certainly cannot do justice to the tremendous pace of research and the spectrum of activity on this important topic, nor does it really relate to the main topic of plant-parasitic nematodes. Notwithstanding, the treatment presented in this book is complete and is relatively concise given the broad nature of the subject. One thing that I found particularly commendable about this book was the lack of detailed descriptions of current control measures for most nematodeplant interactions. Given the tremendous changes currently occurring in this area, a comprehensive rehash would have
Parhtology Publishers:
36 Johnson, A.M. et al. (1987) Exp. Parosrtoi. 63, 272-278 37 Muhlch. M.L. et al. (I 986) Nucleic Acids Res. 14,553 l-5556 38 Tetrlaff CL. et al. (1990) Mel Blochem. Parasitol. 40, I 83 I92 39 Ttipp, CA. et al. (1989) Exp. Parasrtoi. 69, 21 l-225 40 Rapaport, E. et ai. (I 992) Proc. Nat1 Acad. So. USA 89.8577-8580
G. Roman Reddy is at the Department
of
VeterinaryMedione, University ofF\otida, Gainesville, FL 326 I I0880, LISA. Tel: +I 904 392 4700 x5830, Fax: +I 904 392 9704, e-mail: Infectious
Diseases, College of
ROh’[email protected]
been a waste of space and probably out of date by the time this book reaches its intended audience. Instead, most chapter authors confined their remarks to brief summaries and, when appropriate, potential new approaches. In particular, the emphasis on new diagnostic approaches and non-chemical management were appreciated. I found this to be a very useful and complete treatment, especially when considered alongside its companion volume on tropical and subtropical crops2. These two books will provide most scientists with a broad spectrum of information regarding nematode-plant interaction on most of the world’s important agricultural commodities. Although there are some redundancies between the two volumes, they are minor compared to the comprehensive nature of the treatments. These books should be of great use to agricultural nematologists for a number of years to come. References I NlcWe, W.R. (ed.) (1984) Plant and Insect Nematodes, Marcel Dekker 2 Luc, M., Sikora R.A. and Bridge, J. (eds) (I 990) Plant Parasmc Nematodes in Subtropical and Tropical Agncuiture, CAB InternatIonal
Charlie Opperman Department of Plant Pathology Box 76 16, North Carolina State University Raleigh NC 27695-76 16, USA
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Paraatoiogy
Today, vol.
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prepared from conserved regions of the gene*. This is frequently performed for ribosomal RNA genes to accomplish phylogenetic analysis. Generally, parasite genes are cloned based either on their function or the expression pattern of the proteins they encode. However, not all genes can be cloned on the basis of functional properties, since the functions of many genes are unknown. Alternatively, sequencing genes at random allows sequence determination irrespective of their function. The following are some of the commonly employed strategies to clone and sequence parasite genes. Cloning Methods Based on Protein Function Nucieic acid probes. Proteins with similar functions are known to have several conserved regions in their primary structure, which is the basis for isolating genes using heterologous nucleic acids as hybridizing probes. Because of their expected degree of homology, hybridizations with heterologous probes are usually performed under low-stringency hybridization and washing conditions. However, such conditions often result in the cloning of several undesired genes. This method has other constraints: (I) It cannot be used to clone genes unique for a parasite since it is impossible to obtain a probe; (2) a gene of interest must be available from another organism, preferably from a closely related species, to increase the probability of Isolating the gene before initiating the study, and (3) an additional limitation to cloning P. faioparum genes is Its unusual codon usage derived from the parasite’s high A+T content’? As a result, only SIX P. faicrparum genes have been cloned using heterologous probes (see Table I ), Degenerate oiigonucieotldes. Alternatively, protein sequence data can be used to synthesize degenerate oligonucleotides as hybridizing probes. Typically, oligonucleotides are synthesized by back-translating an established protein sequence encoded by the desired gene. However, the protein sequencing depends on the availability of pure protein. Purifying proteins from P. fobparum or other intracellular parasites IS @I995
E swer Sc~enie Ltd 0 I 69-475819 j/$09 50
a very time-consuming and tedious process. To date, aspartyl hemoglobinase is the only protein purified from P. /doparum using conventional purification methods9, which require the propagation of parasites in very large quantities. Subsequently, the sequence data obtained from the purified protein are used to clone the genelo. If sequence information is available from several species for a gene, oligonucleotides can be designed from the conserved regions of the gene. The conserved regions are typically identified by comparing all the sequences. Such oligonucleotides are used as hybridizing probes or PCR primers to clone the gene from another species. The majority of P. folciparum genes are cloned by these methods (Table I). The length and degeneracy of the oligonucleotides play an Important role. Hyde et al.’ I suggest that the optimal length of an oligonucleotide for isolating P. falciparum genes is 35-42 bases. When selecting oligonucleotides, it IS ideal to include amino acids encoded by few codons, in order to minimize the degeneracy. Particularly for PCR primers, the ideal amino acid at the 3’ end of a primer is Met or Trp (each having only one codon), thereby increasing the probability of amplifying the target gene. PCR can also be employed using one degenerate oligonucleotide to the gene of interest to obtain sequence information. In this approach, a library of DNA fragments ligated into a vector are amplified using a degenerate ollgonucleotide to the gene and an oligonucleotide to the vector sequence flanking the gene as PCR pnmers12. Such a method is vet-y valuable for genes in which only one conserved region is identified. However, the major problem in using degenerate oligonucleotides is their false priming to undesired sequences, resulting in the cloning of false positives. False positives can be minimized by performing a second PCR reaction on the fint PCR product using nested primers. Nested primers can be designed from another conserved region internal to the amplified product to increase the specificity of the PCR. functiona/ complementation. Functional properties of gene products are also exploited to isolate genes by genetic 37
Techniques complementation. A functional recombinant protein of a parasite gene can complement a host that lacks the respective functional protein. The genes for citrate synthetase gene of Coxiella burner;; 13, phosphoglycerate kinase of Trypanosoma bruceii4, phosphofructokinase and phosphoenolpyruvate carboxykinase of Hoemonchus contortus Is,’$ triosephosphate isomerase of Giardio /amb/,a17 and glucose phosphate isomerase and ornithine aminotransferase of P. falciporum’* have each been cloned by complementation of E. co/imutants. The adenine phosphoribosyltransferase gene from P. fakiporum has been cloned in mutant mouse L cells that lack the enzyme’9. The major advantage tn this approach is the greater success of cloning a complete, functional gene. However, availability of a suitable mutant in a host is the primary step that often is the major limitation. Antisera. Antisera raised against a protein can be used to isolate the gene encoding that protein from a library constructed in an expresston vector. This approach has two limitattons: (I) large quantities of purified protein are required to raise antisera; and (2) antisera raised against a protein often crossreact with nonspecific antigens. To date, the only metabolic enzyme gene cloned from P. fdciparum by this method is lactate dehydrogenase? Since the major research emphasis on malaria is to develop a vaccine, most of the genes sequenced from P. falciparum encode immunodominant surface antigens. Immune sera from individuals repeatedly infected with malana parasites are used to clone antigen genes, and the majority of antigen genes of P. filciporum are cloned by this method. Nearly 90 different antigen genes/gene fragments have been sequenced, and are listed in the UNDP/World Bank/ WHO /TDR program malaria database available from the World Health Organization. Most of them encode antigens that contain several amino acid repeats. Because of the variation in the repeats2’ of proteins from parasites collected from different locations, such antigens are less likely to be vaccine targets. Thus, the development of alternative methods to clone nonrepetitive antigen genes to which the host develops consistent immunological responses is essential. Cloning Expression
Methods Pattern
I. Plosmodium ~~/C$WWTJ structural
conventional
genes with
known
function
cloned
by
methods
Gene Heterologous Actin
nucleic acid probes
Accession no. a
Year
Ml9146
I988
Calcium ATPase
X7 I 765
I993
Calmodulin
M59770
1991
YOO5I9
I987
P-tubulinb
M3 I205
I989
y-tubulin
X62393
I993
X65738
I993
X61921
1991
JO3028
I987
Hypoxanthine-guanine
phosphoribosyl transferase
Degenerate oligonucleotide Cation ATPase CDC2
probes
protein kinase
Dihydrofolate
reductase-thymidylate
synthetase
DNA
polymerase (Y
L I8785
1991
DNA
polymerase ab
X62423
1991
M24322
I989
X63648
I992
RNA polymerase I
LI II72
I993
RNA polymerase II
Xl6561
I989
RNA polymerase Ill
M37820
1991
Topoisomerase
x79345
I’994
cw-tubulin I
x I 5979
I 989
c-Y-tubulinII
M34390
I 990
P-tubulinb
X I 6075
I 989
Unusual protein kinase
X67288
I 993
Multidrug resistance gene
I (PfMDR I )b
Protein kinase
II
Degenerate oligonucleotide Aspartic hemoglobinase
PCR primers X75787
I 994
Cysteine proteinase
M80590
I992
Dihydroorotate
JO3028
I987
M647 I 5
1991
ERD2
X74869
I993
Hexokinase
M92054
1992
Multidrug resistance gene I (PfMDRl)b
M24850
I989
Multidrug resistance gene 2 (PfMDR2)
M2485 I
1989
3-Phosphoglycerate
DNA
dehydrogenase
polymerase sb
M59249
1991
reductase I
L22057
I993
Ribonucleotide reductase 2
L22058
I993
Triosephosphate
LO 1654.5
I993
LO8200
I993
JO5544
I990
L I 5426
I993
JO3084
I988
M IO985
I985
M I9753
I986
JO4072,3
I988
Heat shock protein
X I5292
I989
Lactate dehydrogenase
Ml4818
I985
X60488
1991
M80655
1991
Xl3014
I988
Ribonucleotide
kinase
isomerase
Vacuolar ATPase Functional complementation Glucose phosphate isomerase Ornithine
aminotransferase
Antisera Aldolase Glycophorin-binding
protein
Heat shock protein 70 (HSP70) Heat shock protein 78 (glucose-regulated
protein)
No methods are described for these genes Elongation factor I (Y Glucose-6-phosphate
dehydrogenase
Heat shock protein 90 (HSP90) a Accession no. and year represent the initial discovery of the gene. b Gene isolated by more than one method.
Based on the of the Genes
Differential screening and subtractive hybridization techniques. Genes expressed in different life cycle stages of a 38
Table
parasite can be cloned by using the differential hybridization screening technique. In this method, labelled cDNA prepared from mRNA of different developmental stages are hybridized sequentially to
a library of the parasite. Clones unique to each stage can then be identified by their differential hybridization patterns. The major advantage of this approach is that it does not requtre prior knowledge Parasitology Today, voi. / I, no. I, I 995
Techniques of the genes. For example, many developmentally regulated genes have been cloned from promastigote/ amastigote stages of Leishmon,o majorZ2 and L. donovc~ni23,and also from Eimetio bovis24.
This method is also appropriate for cloning strain-specific genes. For example, Entomoeba h~stolytica causes amebic dysentery in only 10% of the people infected with this parasite. This is due to variations in the virulence of different parasite strains. By using this method, a strain-specific gene for this parasite has been cloned25. However, this method is only applicable for highly expressed stage- or strain-specific genes. To clone genes expressed at low levels, one needs to utilize additional methods such as the subtractive hybridization technique. In this method, mRNA from one developmental stage is hybridized with cDNA from another stage of the parasite, and the unhybtidized mRNA is separated and used to screen a library. For example, a cyclic AMP-inducible gene from the infective stage of Tryponosoma cruzl is isolated in this mannerz6. Nevertheless, these methods are not very effective for pre-erythrocytic stage genes of P. folciporum, since it is dificutt to isolate mRNA from mosquito or liver-stage parasites that is free from host RNA. Subtractive hybridization technique is also used to clone DNA unique for a genome which is valuable to determine DNA polymorphisms within a parasite species. Representative Difference Anaiysis. Recently, Lisitsyn et a/.17 developed a system, called representative difference analysis, to clone the differences between two complex genomes. In this method, subtractive and kinetic enrichment is used to purify restriction endonuclease-digested fragments unique for a genome. Genomic DNA from two sources with genetic variations are digested with a restriction endonuclease. Then, oligonucleotide adaptors are ligated to one genomic DNA (tester), and annealed to an excess of the other (driver). Subsequently, the DNA is amplified by PCR using oligonucleotlde primers to adaptor sequence. Under these circumstances, DNA fragments unique for the tester DNA are the only ones that contain adaptor sequence on both ends, which allows the exponential amplification of the unique DNA. DNA conserved in both the genomes can only be amplified in a linear fashion, since only one of the two strands can have adaptor sequence on them. Consequently, the exponentially amplified PorositologyToday, vol. I i, no. I, I 995
DNA IS cloned. Such a method may be useful for the rapid identification of regions of DNA linked to genetic rearrangements in parasites. Differential display. Recently, Liang and Pardee2a have developed another method, called differential display, tnvolving PCR that rapidly identifies differentially expressed, low-abundance genes of higher eukaryotes. In this method, mRNA is amplified by using a set of oligonucleotide primers, one primer being annealed to the polyadenylate tail of a subset of mRNA, and the other being short (ten bases) and arbitrary in sequence, so that it anneals at different positions relative to the first primer.. PCR products from different sources are then compared on a DNA-sequencing gel to identify genes expressed differentially. Although not used for parasites, this method may be effective to identify some stage-specific genes. Random Sequencing Approach Expressed sequence tags (ESTs). Recent reports on random sequencing of cDNA libraries from human brainI and Caenorhabditis elegans30 have shown that it is an efficient method for obtaining preliminary data on coding sequences In this method, automated DNA sequencers and computer programs are used to obtain large amounts of sequence information, Recently, we have reported 389 ESTs (GenBank accession numbers T02472-TO2633 and T 17984-T 18255) derived from 550 cDNA clones of the etythrocytic stage of P fabparum3’. Database searches of these sequence tags to nucleic acid and protein sequence databases have led to the putative identification of 90 unique genes (Table 2), of which 28 are similar to previously reported P. folciparum genes. By analyzing the types of genes identified, it is evident that this approach is effective for detecting highly expressed housekeeplng and stagespecific transcripts (Table 2). However, additlonal steps to reduce the clones derived from abundant messages In the library are needed. Such steps will increase the probability of identifying lowcopy, regulatory and stage-specific genes. Another problem with the random cDNA approach is the high redundancy rate (28% In the case of P. f&parum3~). However, this redundancy can be minimlzed by Including addittonal screentng steps to exclude clones for whtch sequence tags are available. Since pure mRNA is not readily available from preerythrocytlc stage parasites, it is not
possible to prepare ESTs from these stages. Gene sequence tags {GSTs). Alternatively, random sequencing of genomic DNA may allow the identification of genes from all life cycle stages. But given that only 60% of the genome of Plasmedium is unique32, the identification of ‘real’ genes is complicated by the presence of non-coding sequence. Recently, we have published another report that describes the strategy by which one can efficiently generate sequence data for P. falciparum genes that overcome some of the problems associated with random sequencing of cDNA and genomic DNA4. In this report, we described the strategy to sequence genomic DNA in order to generate GSTs efficiently. Plasmodium falciparum genomic DNA was digested with mungbean nuclease, which IS used to prepare a plasmid library in E. co/i (mung-bean nuclease cleaves P. fa/c$afum genomic DNA and releases gene fragments with complete or partial coding sequence&s). A large number of recombinant clones were selected at random from this library and sequenced to generate sequence tag data. A total of 673 unique sequence tags were obtained from 400 clones (average length of each tag is -320 nucleotides). These data were reported in the GenBank (accession numbers T02634-TO2808 and TO9496-T09993). The database search of these GSTs revealed the putative identification of 5 I unique genes (Table 3) of which only five encode prev~ously reported P. falciparum genes. The genes identified In this study include proteins expressed in etythrocytic, exoerythrocflic and sexual stages of the parasite. This approach clearly allows the isolation of genes expressed in all stages of the parasite. The PREDICT computer program, based on patterns of codon usage and amino acid composition of P. falciparum genes, identifies open reading frames (ORFs) specific for P. fa/ciparum33. Analysis of GSTs by this program indicated that nearly 90% of the clones with no putative identification have long ORFs. Therefore, the majority of the clones sequenced are likely to be new genes with unknown function. In conclusion, this method offers the promise of obtaining partial sequence data for all the parasite genes at a rapid rate. Although this method is tested only for the P. falciparum genome, it is possible to determine large-scale sequence data for genomes of other parasites by this approach. Genes from many other protozoans, including Trypanosoma34, 39
Techniques Table 2. Plosmodium fakiparum
expressed
sequence tags having significant homology Clone
with database sequences
I
Clone name 01 l4C
Accession no. TO2509
Putative identification 60s acidic ribosomal protein P2
47
0807C
;;8197
Laminin receptor
2
0079c
TO2490
AAC-rich
48
023OC
TO2586
Late-stage histone H3, H4
3
0286C
TO2627
Actin la
49
OIOOC
TO2497
Lipoic acid synthetase
4
0285C
TO2626
Activator
50
0325C
T I8003
Major merozoite
5
Ol47C
TO2534
ADP-ribosylation
51
0682C
Tl8127
Mature-parasite
6
Ol54C
TO2540
Alanine tRNA
52
0412c
T I8048
7
036OC
Tl8018
Aldolase’
Membrane associated Ca-binding protein+
8
016lC
-
Asparagine-rich protein”
53
0676C
Tl8122
Merozoite-specific
9
0283C
TO2625
ATP-dependent
54
0289C
TO2628
Methionyl tRNA
No.
protein I 37 kDa subunit factor 4
synthetase
RNA helicase
No.
name
Accession
.
Putative
identification
surface Ag” infecteda
surface Ag” synthetase
IO
0309c
T I 7990
Breast basic conserved protein
55
Ol3lC
TO252 I
Modifier
II
025lC
TO2605
Calcium-binding protein”
56
0075c
TO2486
Multicatalytic endopeptidase Nucleolar transcription factor
12
0322C
Tl8001
Calmodulin”
57
03ooc
T I 7986
13
01 I IC
TO2506
Casein kinase II alpha
58
OOOIC
TO2472
Nucleolin
14
0706C
Tl8140
CCAAT-binding
59
0327C
T I 8004
ORF
factor
I protein
15
038lC
T I8027
cdc2 I protein
60
0464C
T I8079
Phenylalanyl tRNA
16
0856C
T I8228
Cell division control protein 48
61
08OOC
Tl8193
Pr86 rhoptry-associated protein I”
I7
OlO3C
TO2500
Choline kinase
62
0333c
T I8008
Prohibitin
I8
038OC
T I8026
Circumsporozoite-related
63
0648C
Tl8107
Protein kinase A catalytic chain
I9
0228C
TO2585
Deoxyribonuclease
64
0446C
T I8067
Protein kinase C inhibitor
20
Ol63C
TO2546
Elongation factor I B
65
0757c
Tl8165
RAP-2a
21
OlO5C
T02501,2
Elongation factor 2
66
0224C
TO258 I
RD protein
22
0496C
T18096
Embryo-specific dormancy protein
67
0663C
Tl8l
Rhoptry-associated
23
0787C
Tl8181
Erythrocyte-binding
68
0367C
T I8022
Ribonucleoside-diP reductase
24
0737c
Tl8156
Gene I I-la
25
0792C
Tl8185
Gene fiu
69
0293C
TO2809
(large) Ribonucleoside-diP reductase (small) Ribosomal protein L9
antigen”
proteina
I protein
I5
synthetase
protein
Ia
26
0812C
T I8202
Genes rp0I-f. rpoS
27
05ooc
T I8099
Glucose-6-phosphate
70
Ol95C
TO2564
28
0678C
Tl8124
Glutamic acid-rich protein”
71
049lC
T I8094
Ribosomal protein S9
29
007lC
TO2482
Glyceraldehyde-3-P-dehydrogenase
72
0312C
T I 7993
RNA helicase
30
0675C
T18253
Glycerol-3-P-dehydrogenase
73
0799c
Tl8192
S-adenosyl methionine synthetase
74
0892C
isomerasea
S-antigen precursora
31
Ol65C
TO2548
Glycine tRNA
32
01 l3C
TO2508
Glycogen synthase kinase-3
75
0337c
Tl8012
Sec23
33
0252C
TO2606
Glycophorin-binding
76
0766C
Tl8168
Serine-rich proteina
34
0237C
TO2592
GSTI -HS GTP-binding protein
77
04lOC
T I8047
Serine-rich protein
78
OlO8C
TO2504
Serine-rich protein homologue”
synthetase proteina
35
0709c
Tl8143
Heat shock protein 82
36
Ol57C
TO2543
HGPRT”
79
048lC
T I8087
Splicing factor SC35
37
0418C
T I8054
Histone H2B
80
065OC
Tl8108
Suppressor 2 of zeste
38
076lC
-
Histone H3
81
008OC
TO249 I
T-protein
007oc
TO248 I
TAT-binding
protein
Thioredoxin
precursor
39
0212c
TO2575
HSP70”
82
40
036lC
Tl8019
HSP90a
83
034lC
Tl8015 T18023
Threonyl tRNA synthetase
41
0072C
TO2483
HSF90-B (HSP84)
84
0369C
42
0364C
-
Interspersed repeata
85
Ol85C
86
0789C
Tl8182
Transketolase
87
0795c
Tl8188
Troponin C
88
OIOIC
TO2498
Ubiquitine-conjugating
89
Ol28C
TO25 I9
Vacuolar
ATPase
90
069OC
Tl8130
Vacuolar
ATPase”
43
0323C
T18002
lsoleucyl tRNA
44
0233C
TO2588
Keratin
45
Ol86C
-
Knob-associated protein”
46
0223C
TO2580
histidine-rich
Lactate dehydrogenase”
“Tags having homology to previously reported
protein
enzyme
f. fokiparum genes.
Giardio35, Toxogkwno36, Leishmanio37 and Babesia38,39, have been cloned by using the genease activity of the mungbean nuclease. These reports suggest that such an approach is feasible for these parasites. The major limitation with randomsequencing methods is that It WIII take a long time to identify a specific gene. The probability of isolating a specific gene at random from a P. falciparum gene fragment library is less than one In 40
synthetase
Transformation-sensitive
7500 clones (assuming there are 7500 genes, and all of them are represented only once). However, analysis of our data4 indicates that only one in three clones analyzed have complete coding sequence for genes; the remaining two-thirds have one or more exons. In addltlon, IO% of the clones are redundant clones. These variables reduce the chance of Isolating a gene to less than one out of I4 000 clones sequenced.
Conclusion Approximately 40 metabolic enzyme genes and 90 surface antigen genes of P. folciparum have been isolated by conventional cloning methods. The data obtained by random sequencing methods represent over 800 new genes (of which 108 have significant similarity to genes reported in databases). Most of the data from random sequencing have been obtained, in less than two years’ Parasitology
Today, vol.
I I, no. i, I995
Techniques Table 3. Plasmodium folciparum gene sequence tags having significant homology with database sequences No. I
2 3 4 5 6 7 8 9 IO II I2 13 I4 I5 I6 17 18 I9 20 21 22
TO9726
Putative identification 305 ribosomal protein S- I8 30s ribosomal protein S I2 50s ribosomal protein L I6
TO962 I ,2
605 ribosomal
protein
Ll8A
TO993 I
60s ribosomal
protein
L27a
T09807,8
60s ribosomal
protein
L8
T02745,6
Actin
T09692,3
Adenylyl
TO96 I7,8
Alternative
T09594,5
ATP-dependent
T09922.3
/3 coat protein
T09635.6
Bkm-like sex-determining region hypothetical protein CS3 I9
Clone name 0026M 0368M 0324M 0204M 0482M 0390M 0088M 0284M 0202M 0187M 0470M 021 IM
Accession no. T02656,7 TO978 I ,2
0487M 0075M 0405M 0017M 0362M 0291M 0422M 0431M 0132M 0145M 0136M 0419M 0065M 0421M 0058M 0197M 0225M
T09936.7
Ca’+-dependent
T02726,7
Cathepsin
D (aspartyl proteinase)
T09822,3
Clustered
asparagine-rich
TO2647
Cyclophilin
TO9772
DNAJ protein
T09700, I
DNAJ
T09844,5
DNAJ protein
T09860, I
Duplicate
T02798,9
Dynamin
Ila cyclase gene splicing factor- I RNA helicase
protein
kinase” protein
(Bacillus subtilis) (& cob)
protein
homologue
HSJ I
procyclin
T09530, I
Dynein P-chain
T02796,7
Glucose-6-P
TO984 I
Glutamate
TO271 I.2
Glycerol-3-P
dehydrogenase
T09842.3
GTP-binding
protein
RYH I
TO2702
GTP-binding
protein
YPT3
T09608,9
Initiation
T09654,6
Iron-responsive protein
T09847,8
Lactate dehydrogenase
T09652,3
Liver-stage
32
0424M 0224M 0522M
TO996 I
Mitochondrial protein
33
0175M
TO9580
Mitochondrial pyridine nucleotide transhydrogenase p subunit
34
0314M 0488M 003lM 0158M 0160M
T09709, IO
Nitrogen
T09938.9
PolyA nuclease
T02666,7
Proteasome
TO955 I ,2
Ras-like protein
T09555,6
Regulatory protein cerevisioe
23 24 25 26 27 28 29 30 31
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0188M 0229M 0347M 0337M 0176M 0046M 0201M 0104M 0334M 0167M 0343M 0049M 0543M
“Tags having homology
isomerasea dehydrogenase
factor elF-4A element-binding
antigen (LSA- I)” phosphate-carrier
fixation-U
protein
C3 (Rat) TC4 - Sacchoromyces
T09596,7
Rfbf protein
TO9657
Ribonucleotide
T09752,3
Ring-infected
T09736,7
5. cerevisiae gene (vasa protein)
- 5. typhimurium reductase surface antigen
TO958 I ,2
5. pombe cdc2 I gene
T02688,9
Serine hydroxy
TO96 I5,6
Serine proteinase
T02767,8
Sexual-stage
methyl transferase type
I precursor
specific protein”
T09733,4
Sto~hylococcus xylosus BBM3XM
T09568,9
Thioredoxin
T09744,5
Thrombospondin
T02692.3
Tubulin
T09987,8
Ubiquitin-carrier
to previously
reported
time, by a small group of investigators. Thus, the random sequencing approach provides sufficient sequence information Parasitology Today, voi. i I, no. I, I995
P. fokiparum
precursor
II (c~) protein
genes.
for each gene at a rate that is useful to initiate a detailed study. Numerous new avenues of research into the bio-
chemistry of the parasite have been opened using the genes identified to date by random sequencing approach, which may lead to targeted drug development. The sequence tags with no putative identification are also valuable resources for initiating studies to Identify new antigen genes. The sequence data from the random sequencing project are recorded in the Genbank database and the clones are readily available to initiate such studies. In addition, these tags can serve as a valuable tool in recent efforts40 to construct fine resolution maps of P. folciparum chromosomes. In summary, a gene of interest can be cloned by one or more methods as described in this review. Isolation of a specific gene is easier by conventional methods compared to the random sequencing approach. However, the random sequencing approach provides the opportunity to obtain sequence information of all the genes of a parasite such as P. faiciparum, in three to five years. This should lead to the development of large-scale basic and applied research programs aimed at developing strategies for both chemotherapeutic and immunological control. Acknowledgements I wish to thank Ky Minh
Tran and Suhasin Ganta for thelr assistance In preparing this manuscript. My thanks are also extended to Ray Kaplan, Debopam Chakrabartl, John B. Dame and David R. Allred for their helpful discussions. This work was supported by the Univenity of Flonda, Gainesville, USA.
References
I Weber, J.L. ( 1988) Exp. Porosrtoi. 66, I43 I70 2 Pollack, Y. et a/. (I 982) Nucierc Acids Res IO, 539-546 3 McCutchan, T.F. et al. (I 984) Scrence 225, 625-628 4 Reddy, G.R. et al. (I 993) Proc. Notl Acad Scr USA 90,9867+987 I 5 Vemtck, K.D. et ai (I 988) Nucleic Acids Res. 16, 6883-6896 6 T&x, T. and Kemp, D.J. (I 99 I) Mol. Blochem. Porawtol. 44, 207-2 I2 7 de Bran, D. et 01 (I 992) Genomlcs 14, 332-339 8 Wllks, A.F.( 1989) Proc Not1 Acod So USA 86, I603-l 607 9 Goldberg, D.E. et oi. (199l)j. Exp. Med. 173, 96 l-969 IO Francis, S.E. et 01. (I 994) EMMBOJ. 13, 306-3 I7 I I Hyde, J.E. et al. (I 989) Mol. Biochem. Parosrtoi. 32, 247-262 I2 Oberle, 5.M and Barbet, A.F. (I 993) Gene I36, 29 I-294 13 Heinren, R.A. and Mallavla, L.P. ( 1987) In@ct. Immun. 55, 848-855 I4 Alexander, K. et 0). ( 1990) Gene 90, 2 15-220 R.D. et 41. (199 I) Mol. Blochem. I5 Klein, Parasrtol. 48, 17-26 R.D. et 01. (1992) MO\ Blochem. I6 Klein, Porasrtoi 50, 285-294 17 Mowatt, M.R. et aI (1994) txp Porosltoi 78, 85-92 41
Techniques (I 990) J. 5101.Chem. 265, 12337-12341 Pollack Y. et al. (I 985) Exp. Parasftoi. 60. 270-275 Simmons, D.L. et 01. (I 985) Mol. Blochem. Poras,tol. 15, 23 I-243 Kemp, D.J. et 01. (I 990) Adv. Parasrtol. 29, 75-149 Coulson, RM.R. and Smith, D.F. (I 990) Mol. 6,ochem. Parasrtoi 40, 63 -76 Joshl, M. et al. (1993) Mol. Bfochem. Parasltoi. 58,345-354 Abrahamsen, MS. et al. (I 993) Moi Blochem. Parasitol. 57, I ~ I4 Burch, D.J. et al. (I 99 I) 1, C/in. Mlcrobiol. 29, 696-70 I Heath, S. et al. (I 990) Moi Biochem. Parosrtoi. 43, 133-142
I8 Kaslow, D.C. and Hill, S. 19 20 21 22 23 24 25 26
Plant Parasitic Nematodes in Temperate Agriculture edited by
K. Evans, D.L. Trudgill and
JM. Webster, CAB International, 1993. f75.00 {xi + 648 pages) ISBN 0 85 I98
808 3
Plant nematology has changed dramatically during the ten yean since the last comprehensive book cataloging plantnematode interactions was published’. Many chemical-control practices for nematode pests have either been eliminated or severely curtailed in thetr application due to increased environmental and health awareness. In addition, intensive agricultural production systems have exacerbated existing nematode problems and resulted in the identification of new interactions. As crop damage due to plant parasitic nematodes has increased, funding for research and development of new control practices has dwindled. In spite of this last problem, the past decade has been a period of tremendous excitement and promise in several areas of plant nematode research. It is quite appropriate that these advances be incorporated into a generally available and accessible form. This book provides a comprehensive look at plant-parasitic nematodes attacking a wide range of crops grown in temperate climates, and is a companion volume to the previously released treatise on tropical agriculture2. As such, these two volumes provide an overview of the current status of plant parasitic nematode damage and control in world agricultural systems. The I7 chapters in this book cover both major and minor crops grown in temperate climates, including omamentals, bulb crops, glasshouse crops, mushrooms, and forest trees. Most of the listed commodities 42
27 Lisitsyn, N., Lisitsyn, N. and Wigler, M. (I 993) Soence 259, 946-95 I 28 Liang, P. and Pardee. A.B. (I 992) Science 257, 967-97 I 29 Adams, M.D. et al. (1992) Nature 335, 632-634 30 Waterston, R. et al. (I 992) Nature Genet. I, 114-123 31 Chakrabatii, D. et a/. (1994) Mol. Blochem. Parasitol. I 66, 97-l 04 32 Dore, E. et al. (I 980) Mol. Biochem. Parasitol. I, 199-208 D. (1988) Mol. 33 Saul, A. and Battistutta, Blochem. Parasitol. 27, 35-42 34 Brown, K.H. et al. (1986) J. Bioi. Chem. 261, 10352-10358 35 Adam, R.D. et al. (I 989) 1. Exp. Med. 167, 109-l I8
have been overlooked in previous books in favor of the more traditional agricultural crops, but given the changing nature of production in the 1990s it is appropriate that they are Included here. Of course, the major row and vegetable crops are also covered in this book In addition to the chapters on nematodecrop interaction, there are treatments on nematode extraction and identification, nematode population dynamics and yield loss, molecular diagnostics, quarantine, molecular approaches to control, and insect parasitic nematodes. These are all well-written and useful treatments (particularly the extraction and population dynamics chapters), but the lone chapter on insect nematodes seems a bit out of place in this particular venue. One chapter certainly cannot do justice to the tremendous pace of research and the spectrum of activity on this important topic, nor does it really relate to the main topic of plant-parasitic nematodes. Notwithstanding, the treatment presented in this book is complete and is relatively concise given the broad nature of the subject. One thing that I found particularly commendable about this book was the lack of detailed descriptions of current control measures for most nematodeplant interactions. Given the tremendous changes currently occurring in this area, a comprehensive rehash would have
Parhtology Publishers:
36 Johnson, A.M. et al. (1987) Exp. Parosrtoi. 63, 272-278 37 Muhlch. M.L. et al. (I 986) Nucleic Acids Res. 14,553 l-5556 38 Tetrlaff CL. et al. (1990) Mel Blochem. Parasitol. 40, I 83 I92 39 Ttipp, CA. et al. (1989) Exp. Parasrtoi. 69, 21 l-225 40 Rapaport, E. et ai. (I 992) Proc. Nat1 Acad. So. USA 89.8577-8580
G. Roman Reddy is at the Department
of
VeterinaryMedione, University ofF\otida, Gainesville, FL 326 I I0880, LISA. Tel: +I 904 392 4700 x5830, Fax: +I 904 392 9704, e-mail: Infectious
Diseases, College of
ROh’[email protected]
been a waste of space and probably out of date by the time this book reaches its intended audience. Instead, most chapter authors confined their remarks to brief summaries and, when appropriate, potential new approaches. In particular, the emphasis on new diagnostic approaches and non-chemical management were appreciated. I found this to be a very useful and complete treatment, especially when considered alongside its companion volume on tropical and subtropical crops2. These two books will provide most scientists with a broad spectrum of information regarding nematode-plant interaction on most of the world’s important agricultural commodities. Although there are some redundancies between the two volumes, they are minor compared to the comprehensive nature of the treatments. These books should be of great use to agricultural nematologists for a number of years to come. References I NlcWe, W.R. (ed.) (1984) Plant and Insect Nematodes, Marcel Dekker 2 Luc, M., Sikora R.A. and Bridge, J. (eds) (I 990) Plant Parasmc Nematodes in Subtropical and Tropical Agncuiture, CAB InternatIonal
Charlie Opperman Department of Plant Pathology Box 76 16, North Carolina State University Raleigh NC 27695-76 16, USA
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