Initial characterization of several actin genes in the parasitic nematode, Ascaris suum

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INITIAL

CHARACTERIZATION PARASITIC Michael

Wayne Received

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May

11,

OF SEVERAL ACTIN GENES NEMATODE, ASCARZS SUUM A. Winrowl

578-585

IN THE

and Ann Sodja*

Department of Biological State University, Detroit,

Sciences Michigan

48202

1991

Southern hybridization data suggest a large actin gene family in Ascaris suum. Our genomic reconstruction experiment indicated that it consists of 50-75 members. Polymorphism was uncovered in the actin genes or in their surrounding sequences. From the genomic library 5 nonoverlapping actin clones were isolated and characterized. 0 1991Academic Press, II

Actins are well conserved through evolution at both the amino and nucleic acid sequence levels (1,2). With few exceptions, they are encoded by a multigene family, members of which appear expressed in a spatially Great variations exist between and temporally specific manner (3-6). organisms with respect to actin gene number, structure, and organization on the genome (7). In vertebrates the actin-associated 3’ transcribeduntranslated regions (UTRs) are unique to the gene encoding a particular actin isotype, are conserved in orthologous fashion between several vertebrate species diverge at rates lower than expected for noncoding DNA; hence they may, and do in some instances, play a role vis a vis actin gene expression (8,9). We chose to investigate actin genes in the parasitic nematode, Ascaris suum. To date the only nematode in which these were studied is Caenorhabditis elegans. The present study reports the isolation and partial characterization of A. suum actin genes.

IPresent Michigan *

address: Department of Microbiology State University, East Lansing, Michigan

To whomcorrespondence should be addressed.

DOO6-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. 411 rights of reproduction in any form reserved.

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of tissues and DNA

isolations.

A. suum adults were collected from Frederick and Herud Meat Packing Company in Detroit, MI. Eggs were removed from uteri and allowed to develop to first larval stage. The larval chitinous layer was removed (lo), the eggs homogenized in DNA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 100 mM EDTA). Their DNA as well as DNA from adult worms was isolated using CsCl gradients (11). Southern and dot-blot hybridizations. Genomic DNAs were digested with several restriction endonucleases and analyzed by Southern hybridizations (12). Probes used were a 32P-labeled 0.8-kb Sal1 fragment, with a partial actin coding sequence from the act5C gene of Drosophila melanogaster (Dm probe) (6), or a 1-kb EcoRI/BamHI fragment, containing one of the 4 actin genes of C. elegans (Ce probe) (13.14). For dot-blot hybridizations, an A. suum actin clone, pAsA4E, was digested with EcoRI and serially diluted to mass amounts equivalent to the different copy numbers that may exist in the post-diminutive haploid genome. Using the protocol of Schleicher and Schuell, the DNA solutions were applied to a nylon filter, used in Southern hybridization (12). Quantitative analysis of the resulting autoradiograms was done in a Beckman DU-8 spectrophotometer at 500 nm. Construction of Genomic Library. The cloning of genomic DNA from 12-day larvae into hEMBL-3 (Promega Biotec) was according to Frischauf et al., (15).

Isolation and restriction containing recombinant phage.

enzyme

mapping

of

actin

The recombinant library was amplified and screened with the Dm actin probe. The DNAs from the plaque purified recombinant phage were isolated, and low resolution restriction enzyme maps were generated (16).

RESULTS Copy

number

of actin

genes.

Our own, as well as previously reported Southern hybridization data (17,18), of pre- and post-diminutive DNAs suggested that A. suum has multiple actin genes, none of which appear to be lost during chromosomal diminution. In our experiments the Dm and Ce probes resulted in identical hybridization patterns, confirming Because the number of the high evolutionary conservation of actin genes. genomic DNA fragments visualized in Southern hybridizations cannot be a priori equated to the number of genes, a quantitative dot-blot genomic reconstruction experiment was performed. Fig. 1 Al shows the autoradiogram of the reconstruction dot-blots and A2 the hybridization between the Dm probe and A. suum genomic DNA. The genomic dots and the copy number equivalents indicated haploid genome. between 50-75 actin genes per A. suum post-diminutive In panel Bl, dots 1 and 2 contain PBS(+) and $X174 DNAs, respectively, as 519

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The DNAs Dot-blot genomic reconstruction experiment. 1. dot-blotted on nylon filter and probed with denatured, neutralized, 32P-labeled D M actin probe. Exposure was 24 h without intensifying In lane 1, the 4.3-kb screens. A. A. suum reconstruction experiment.

Fig. were

EcoRI fragment from pAsA4E was dot blotted in mass amounts equivalent to 1, 2, 4, 8, 16, 24, 32, 40, 50, 75, and 100 actin genes per haploid genome. Lane 2 contained 0.1, 0.5, 1.0, and 2.0 pg of A. suum somatic DNA. B. Control dot-blot. Fifty-eight ng of PBS(+) and 63 ng 9x174, respectively, were dot-blotted and hybridization

Prehybridization to detect non-specific hybridization. conditions are described in Materials and Methods.

controls for nonspecific intensities observed in sequences alone.

Actin

gene

hydridization. None was detected, hence the panel Al reflect hybridizations due to actin

polymorphism.

Restriction fragment length polymorphism (RFLP) can result from either a base change at a restriction site or from variability in the number of repetitive sequences in a To determine whether polymorphism is associated restriction fragment. with the A. suum actin genes, DNAs from individual and a pool of worms were analyzed. The two hybridization bands in the 9.4-kb region and at least 5 major actin hybridizing fragments between 6.7- and 2.0-kb size are present in all DNAs (Fig. 2). However, individual 10 lacks a 6.3-kb restriction fragment conserved in all others. It appears to be supplanted by a doublet consisting of 5.9- and 5.4-kb fragments. The individuals in lanes 3, 6, 7, and 10 posses a 1.35-kb actin fragment, not detected in the others. A 7.8-kb fragment was not detected in individuals 2 and 3. At least five different hybridization patterns were observed (Fig. 2, lanes 1, 2, 7, 9, and 10). Restriction enzyme mapping. From a genomic underrepresented amplified library, 11 isolates were obtained; 5 of these contain unique, nonoverlapping actin-containing DNA fragments. Their restriction maps are shown in Fig. 3. The actin-hybridizing fragments range in size from 580

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Fie. 2. Restriction fragment length polymorphism. EcoRI digested DNAs isolated from internal organs of 10 different A. suum individuals (lanes l-10) and from a pool (lane P) were hybridized with 32P-labeled D m actin probe as in Fig. 1. The autoradiographic exposure was for 8 d with two intensifying screens. The arrows indicate areas of polymorphic variation. The size standard used was Hind111 digested h and @x174 DNA.

about 2- to 8kb within the insert DNAs of 13.8- to 17.2-kb. No conserved restriction sites with respect to the endonucleases used were identified that they are derived from between these recombinant phage, suggesting different non-overlapping loci. The intensities of hybridization between the A. suum DNA fragments and the D m probe were virtually identical, suggesting that none of the recombinants contain more than 1 actin gene. A 0.7-kb BamHI/SmaI fragment in hAsA did not hybridize with the D m No obvious similarities were detected between these maps and probe. those of the 4 Ce actin genes.

DISCUSSION Our Southern hybridization analyses of A. suum pre- and postdiminutive genomic DNA and those previously reported (17), indicate that a large gene family and that no actin actin genes in A. suum comprise genes appear to be lost during chromosomal diminution. During this event, the bulk of the DNA lost is comprised of satellite sequences; however, some low and single copy interspersed sequences which may represent structural genes are lost as well (19,20). In a large gene family such as 581

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Fig. 3. Low resolution restriction enzyme maps. One pg of DNA from the 5 recombinant phage was used in single and double digestions with restriction endonucleases, BamHI, EcoRI, SalI, and SmaI. Absence of any one of these enzymes indicates that there was no restriction site within the cloned sequences. A 32P-labeled Dm actin probe was used. The symbols identifying the coding, noncoding and h arms are indicated.

actin, however, the loss of a few members probably would not be detected in a Southern hybridization analysis. The results from the reconstruction experiment indicated that A. suurn has 50-75 actin gene sequences, in contrast to the 4 found in C. elegans or in other nematodes studied (21). The only reported precedents to this high copy number are for humans, mice, and petunias (22-24). The multiple actin genes in A. suum may assure the synthesis of actin in amounts required during its life cycle and/or allow for differential expression of individual genes. Also many of these actin gene sequences may be pseudogenes. RLFPs were observed among the actin genes in sea urchins, sea star, and humans (25-27). Southern blot hybridizations of the EcoRI digested DNA revealed that most of the actin hybridizing fragments were present in all individuals. However, the 5 different hybridization patterns observed indicated RLFPs among the A. suum actin genes. Other restriction endonucleases, gene-specific actin probes or lower hybridization stringencies may reveal more RFLPs (26). Five unique non-overlapping actin-containing h recombinants were isolated and their characterization initiated. The hybridization intensities 582

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in each of the 5 isolates were identical, regardless of the size of the actin hybridizing fragment (2=8-kb), suggesting that each recombinant contains one actin gene. Thus, the coding regions of these 5 actin genes are at least 13.8- to 17.2-kb apart. This is in contrast to C. elegans in which 3 of the 4 actin genes are located on a 12-kb genomic DNA fragment (28). Because there exist many actin gene sequences in A. suum, it is more than likely that some of them are closely linked on the genome. The restriction maps of the 5 A. suum recombinant phage revealed no obvious similarities to those of C. elegans actin genes. All 4 Ce actin genes contain at least 2 introns of about 55 bp, except for one of about 2000 bp. In hAsA the 0.7kb BamHI/SmaI fragment may represent an intron or a more divergent actin coding sequence unable to form a stable hybrid under the hybridization conditions. If indeed it is an intron, it would represent a large intron when compared with most of those in C. elegans actin genes. In vertebrate actins, the 3’UTRs are evolutionarily conserved and for some a regulatory function in actin gene expression demonstrated (9). No extensive information regarding these regions exists for invertebrate actin genes. However, one of the actin 3’UTRs in C. elegans may regulate steadystate levels of the transcript (29). Hence, the 5’ A. suum actin recombinants and Eco RI digested genomic DNA were hybridized with each of the 4 Ce 3’UTR probes. No cross-hybridization of these sequences between the two nematodes was detected (data not shown). If the matched sequences are short and scattered throughout a stretch of DNA, stable hybrids may not form under the conditions used. If the lack of sequence similarities in the 3’UTRs of C. elegans and A. suum is confirmed by direct sequencing, the results would represent a departure from the observed interspecies sequence conservation in the 3’UTRs of orthologous vertebrate actin genes. It is anticipated that the data obtained from a more detailed characterization of the actin genes isolated, combined with the existing data, will contribute to the current understanding of molecular evolution of eukaryotic gene structure and regulation.

ACKNOWLEDGMENTS We are grateful to Dr. Michael Krause for the Ce probes. We thank Dr. Mark Weiss and Timothy Hadden for helpful discussions and critical evaluation of the manuscript. This work and M.A.W. were supported by the NIH-GM08167 grant. Further support was provided by a grant from the Center for Molecular Biology at Wayne State University.

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