©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of the Gene Encoding Nuclear DNA Helicase II
A BOVINE HOMOLOGUE OF HUMAN RNA HELICASE A AND DROSOPHILA Mle PROTEIN (*)

Suisheng Zhang (§) , Heiko Maacke , Frank Grosse (§)(¶)

From the (1)Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Martinistrasse 52, D-20251 Hamburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nuclear DNA helicase II (NDH II) unwinds both DNA and RNA (Zhang, S., and Grosse, F.(1994) Biochemistry 33, 3906-3912). Here, we report on the molecular cloning and sequence determination of the complementary DNA (cDNA) coding for this DNA and RNA helicase. The full-length cDNA sequence was derived from overlapping clones that were detected by immunoscreening of a calf thymus cDNA library in bacteriophage gt11. This cDNA was 4,528 bases in length, which corresponded well with a 4.5-4.7-kilobase-long mRNA as detected by Northern blot analysis. The open reading frame of NDH II cDNA predicts a polypeptide of 1287 amino acids and a calculated molecular mass of 141,854 daltons. NDH II is related to a group of nucleic acid helicases from the DEAD/H box family II, with the signature motif DEIH in domain II. Two further proteins of this family, i.e. human RNA helicase A and Drosophila Maleless (Mle) protein, were found to be highly homologous to NDH II. With RNA helicase A, there was 91.5% identity and 95.5% similarity between the amino acid residues; with Mle protein, we observed a 50% identity and an 85% similarity. Antibodies against human RNA helicase A cross-reacted with NDH II, further supporting that NDH II is the bovine homologue of human RNA helicase A. Immunofluorescence studies revealed a mainly nuclear localization of NDH II. A role for NDH II in nuclear DNA and RNA metabolism is suggested.


INTRODUCTION

Nucleic acid helicases unwind double-stranded DNA and RNA coupled with the hydrolysis of a nucleoside 5`-triphosphate for energy supply. Numerous processes of nucleic acid metabolism including DNA replication, DNA repair, DNA recombination, transcription, RNA splicing, and translation are aided by DNA and/or RNA helicases. These enzymes play a key role in resolving secondary structures to expose single-stranded templates for various DNA or RNA polymerases to copy on, to facilitate the binding of sequence-specific regulatory proteins, to reorganize the nucleoprotein complex, and to mediate sequential dis- and reassembly of higher order nucleic acid structures for the expression of their destined functions (for reviews, see Refs. 1-4). On the basis of computer-assisted amino acid comparisons, a so called superfamily of helicases has been found that contains common helicase domains, including a nucleotide-binding motif(5) . The members of this protein family can be subdivided into superfamily I and superfamily II according to their different sequence signatures(5) . A DEAD/H motif found in domain II defines an expanding group of DNA and RNA helicases from superfamily II(6, 7, 8) , which include proteins with important regulatory functions in several pathways of genome expression.

We have recently purified and characterized two DNA helicases from calf thymus nuclei, designated as nuclear DNA helicase I (NDH I)()and nuclear DNA helicase II (NDH II) (9). Homogeneous NDH II displays a rather high molecular mass of around 130-140 kDa. While this enzyme was initially identified as DNA helicase, it was subsequently shown to be associated with an RNA-dependent NTPase and to be able to unwind double-stranded RNA(10) . Here, we report on the sequence of the NDH II-encoding cDNA. The derived amino acid sequence of bovine NDH II turned out to be 91.5% identical and 95.5% similar to that of human RNA helicase A(11) , an enzyme previously identified as RNA helicase only(12) . Furthermore, there was a 50% amino acid identity, and a 85% similarity with the Maleless protein (Mle) of Drosophila(13) , suggesting that NDH II is the bovine homologue of these two proteins. Combining data from sequence analysis and biochemical properties, Mle proteins might be considered as DNA and RNA helicases that are required for the regulation of both DNA and RNA secondary structure in gene expression and perhaps also in DNA replication.


EXPERIMENTAL PROCEDURES

Polyclonal Antibodies against NDH II

400 µg of pure NDH II was injected subcutaneously into a rabbit. After 2 weeks, a second injection was performed with 200 µg of protein, followed by two further injections of 200 µg (each) after another 4 and 8 weeks. Rabbit antiserum was collected 7-10 days after each injection. Quantity and specificity of the antibodies were examined by dot-blot assays. Serum harvested after the fourth immunization (10 weeks after the first injection) was used for library screening, Western blotting, and immunofluorescence studies.

Isolation and Sequencing of cDNA Clones Encoding NDH II

An oligo(dT)-primed gt11 library, constructed from calf thymus poly(A)-containing RNA, was purchased from Clontech. About 210 recombinant phages were screened with the antibodies described above by following standard procedures(4) . As many as 40 colonies producing immunopositive signals were identified after the initial round of library screening. Thereof, 20 colonies were selected for further purification by two successive rounds of antibody screening. Insert sizes of positive clones were determined by PCR using 5`-GGTGGCGACGACTCCTGGAGCCCG-3` as forward primer and 5`-TTGACACCAGACCAACTGGTAATG-3` (flanking the EcoRI site of the gt11 vector) as reverse primer(15) . Clone 6, containing 2168 bp, and clone 12, containing 1419 bp (Fig. 1), were selected and subcloned into the EcoRI-digested vector M13 mp18. Nucleotide sequences of the cDNA inserts on both strands from the M13 mp18 vector were determined by Sanger's dideoxy method(14) . 7-Deaza-dGTP (Boehringer Mannheim) was used to eliminate band compression effects.


Figure 1: Strategy for cloning the cDNA of NDH II. The relative positions of the cDNA subclones, PCR primers, and the PCR probe are indicated. For more details, see text.



The remaining immunopositive clones were PCR-screened by using the 24-mer oligonucleotide 12-F-6 (Fig. 1) as forward primer and a reverse primer from gt11. By using this method, clone 7 was found to extend most to the C terminus. The 3.3-kb insert of clone 7 was subcloned into M13 mp18 and sequenced (Fig. 1). From this a coding length containing 110 amino acids downstream of clone 6 cDNA was obtained. Because we did not obtain full-length cDNA by antibody screening, we rescreened the cDNA library with a PCR probe (300 bp in length) that was synthesized from a downstream part of clone 7 (Fig. 1). About 310 phages were screened with the randomly labeled PCR probe. Nucleic acid hybridization and detection of the clones were achieved by standard procedures(14) . In total, 20 positive clones were obtained by using this method. To identify the clones covering the missing sequence at the C terminus of the reading frame, PCR characterization of cDNA inserts was performed as described above, using the insert-specific primer 7-F-2 (Fig. 1), combined with one of the gt11 vector forward and reverse primers, to account for cases where cDNA inserts might have been oriented in both directions. In this way, two clones, clone 4 (1450 bp) and clone 10 (2150 bp), both of which provided the longest extensions over the 3` terminus of clone 7, were subcloned and sequenced.

The 5`-terminal sequence of NDH II cDNA was obtained by using a 5`-anchored PCR method(16) . 1 µg of calf thymus total RNA, prepared by the guanidinium isothiocyanate/acidic phenol method(17) , was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) and the 21-mer antisense primer A1 (5`-GCCTTAGCATTTTCCAAGGTC-3`, see Fig. 1) that was complementary to a region at 460 bp downstream of the 5`-end of clone 6. After removal of excess primer by GlassMAX spin cartridge (Life Technologies, Inc.), the first-strand cDNA was tailed with dGTP in a terminal transferase-catalyzed reaction(18) . The 5`-anchored PCR product was obtained by amplifications with two anchor primers, ANpolyC (5`-GCATGCGCGCGGCCGCGGAGGCCCCCCCCCCCCCC-3`) and AN (5`-GCATGCGCGCGGCCGCGGAGGCC-3`) at a ratio of 1:9, and the second internal cDNA primer A2 (5`-GGGTCGACTTTCAAGGGTCGCTTGGACTT-3`) (Fig. 1) containing a SacII and a SalI restriction site (underlined), respectively. Southern blot hybridizations were performed to examine the successful synthesis of first-round PCR products with the third internal primer A3 (5`-ATATCCTTCGACGCGAACCT-3`) that was 5`-labeled with [-P]ATP. Hybridized DNA bands were identified on 1% agarose gels at lengths between 650 and 700 bp. 1-5 µl of agarose, containing DNA bands of this length, were transferred to a PCR reaction mixture for the second round of amplification, again with primers AN and A2. The second round PCR products were sequentially digested by SacII and SalI and were subsequently cloned into the plasmid vector Bluescript SK+ II and transformed into the Escherichia coli strain XL-Blue (Stratagene). Colonies carrying NDH II-5`-anchored inserts were screened with the radiolabeled primer A3 (see above). Plasmid DNA was prepared by alkaline lysis followed by ultracentrifugation through a CsCl gradient(14) . Purified plasmid DNA was denatured by alkali and sequenced in both directions with M13 universal primers and sequence-specific primers.

Northern Blot Analysis

Enrichment of calf thymus poly(A) RNA from total RNA was achieved by using oligotex-dT mRNA spin columns obtained from Qiagen. 2.5 µg of poly(A) RNA was electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde and transferred onto a Nitran membrane (Schleicher & Schuell). The membrane was baked at 80 °C under vacuum for 2 h. Prehybridization was performed by incubating the membrane in a roller bottle with 5 ml of 5 SSC (1 SSC contained 0.15 M NaCl, 0.015 M sodium citrate), 2 Denhardt's solution (1 Denhardt's solution contained 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.1% SDS, and 100 µg of salmon sperm DNA per ml for 2 h at 65 °C. Hybridization was performed by incubation for 16-18 h at 65 °C with an NDH II-specific PCR probe. The probe, spanning clone 6 from nucleotides 1117 to 2012, was labeled to a specific radioactivity of 110 cpm/ml by using random primers and [-P]dCTP. Following hybridization, the membrane was washed twice for 15 min each at room temperature in 2 SSC and 0.1% SDS, followed by washing for 15 min at 65 °C in 1 SSC and 0.1% SDS and 30 min at 65 °C in 0.1 SSC and 0.1% SDS. Autoradiography was performed at -80 °C for 3 weeks.

Western Blot Analysis

Calf thymus nuclear extract was prepared as described(9) . 10 µl of nuclear lysate at a concentration of 9.45 mg of protein/ml and 0.5 µg of pure NDH II were electrophoresed through a 7.5% SDS-polyacrylamide gel (19) and transferred onto a Hybond-C extra membrane (Amersham Corp.) by using a semi-dry electroblotter. The membrane was incubated with rabbit polyclonal antibodies against NDH II and human RNA helicase A (kindly donated by Dr. J. Hurwitz, Memorial Sloan-Kettering Cancer Center, NY) at a dilution of 1 to 1000. The primary antigen-antibody complexes were visualized by an enhanced chemiluminescent (ECL) immunodetection procedure as recommended by the manufacturer (Amersham).

Subcellular Localization of NDH II by Immunofluorescence Microscopy

Monkey TC-7 kidney cells were adhesively grown to subconfluence on coverslips within cell dishes. The coverslips were removed and rinsed for several times with PBS (PBS contained 10 mM sodium phosphate, pH 7.5, 140 mM NaCl, 3 mM KCl), air dried, and then fixed with acetone for 10 min at room temperature. Following removal of residual acetone by air drying, the cells were briefly washed with PBS. Acetone-fixed cells were incubated for 1 h with anti-NDH II serum at room temperature. The optimal dilution of antiserum was experimentally determined as 1:700 in PBS. After several washes with PBS, the cells were incubated for another 1 h at room temperature with biotinylated anti-rabbit IgG antibody (Amersham), diluted 1:200 with PBS. The cells were washed and subsequently stained with fluorescence dye-conjugated avidin (Cy3-conjugated avidin; Dianova, Hamburg) that was diluted 1:400 in PBS. Finally, the cells were washed and prepared for microscopy by adding a drop of mounting medium (10% glycerol, 5% polyvinyl alcohol in PBS) and topping them with a thin coverslip. The cells were viewed with a Zeiss fluorescence microscope. Antibody-depleted control serum was prepared by incubating 1:700-diluted anti-NDH II serum with an excess of purified NDH II.


RESULTS AND DISCUSSION

Cloning and Sequencing NDH II-encoding cDNA

The total cDNA sequence of NDH II was obtained by immunoscreening of a gt11 cDNA library of calf thymus poly(A) mRNA; the very 5` and 3` termini of the cDNA were determined by using PCR approaches. Combining the nucleotide sequences from several overlapping clones resulted in a cDNA of 4528 bp in length (Fig. 1). Between the 141-bp-long 5`-untranslated region and the 523-bp-long 3`-untranslated region, NDH II-encoding cDNA contains an open reading frame of 3864 bp. The open reading frame starts with the initiation codon ATG at nucleotides 142-144 and ends with the stop codon TAA at nucleotides 4003-4005. It encodes a polypeptide of 1287 amino acids and a predicted molecular mass of 141,854 daltons. A purine-rich, putative ribosomal binding site (5`-GAAGAAGA-3`) could be identified at the 5`-untranslated region prior to the initiation codon at nucleotides 124-131; four polyadenylation signals (5`-AATAAA-3`) were found downstream of the stop codon at nucleotide positions 4042-4047, 4192-4197, 4290-4295, and 4457-4462.

NDH II Is the Bovine Homologue of Human RNA Helicase A and Drosophila Maleless Protein

The predicted protein sequence of NDH II was used for searching similar sequences in several data bases, such as the SwissProt, the PIR, and the GenBank/EMBL data base, by using the FASTA computer program. The NDH II protein sequence displayed high scoring indexes with two proteins, namely human RNA helicase A (11) and Drosophila Maleless protein (Mle)(13) . With RNA helicase A there was a 91.5% identity and 95.5% similarity between the amino acid residues (Fig. 2A); with Mle protein we observed a 50% identity and an 85% similarity (Fig. 2B). All three proteins belong to superfamily II of DNA and RNA helicases(5, 6, 7, 8) . Members of this family contain six highly conserved amino acid domains and the signature motif DEXH. Bovine NDH II contains the conserved domains within 353 amino acids spanning from residue 411 to 764 (Fig. 3). Here, domain I contains the putative Walker-type nucleotide binding site(20) , consisting of motif A as GCGKT and motif B as FILDD (Fig. 3). In domain II, all three helicases show the DEXH signature motif as DEIH (Fig. 3). In domain VI, the motif QRKGRAGR is strongly conserved between NDH II, Mle, and RNA helicases A (Fig. 3) as well as the vaccinia virus-encoded RNA helicase nucleic acid-dependent phosphohydrolase II(21) . This so-called HRIGRXXR region has been shown to be involved in RNA binding and RNA-dependent ATP hydrolysis of the eukaryotic translation initiation factor eIF-4A (22). Within the six conserved domains, NDH II displays weak but still detectable similarities to three putative RNA helicases from yeast, i.e. the splicing factors PRP2(23) , PRP16(24) , and PRP22 (25).


Figure 2: Protein matrix comparisons between NDH II and RNA helicase A (A) and between NDH II and Mle protein (B). The comparison was made by using DNA Strider software (38) with a window of 23 and a stringency of 7 amino acids. The identity and similarity between NDH II and RNA helicase A was 91.5 and 95.5%, respectively, and that between NDH II and Mle protein was 50 and 85%, respectively. Dots in the lowerrightcorner of each plot represent the 16 imperfect amino acid repeats GGG(G/D)(Y/V)(G/S/V/R)GG.




Figure 3: Schematic representation of NDH II. A, depicted are sequence motifs that have been also found in other proteins. dsRBD I and dsRBD II represent double strand RNA binding domains I and II, NTP represents the Walker-type nucleotide binding domain, DEIH is the so called DEAD/H box motif of helicase superfamily II, NLS stands for a putative nuclear localization sequence, NA bdg. represents a conserved nucleic acid binding domain, and G-rich mirrors the location of the glycine-rich C-terminal repeats. Furthermore, the relative positions of the six conserved domains common to helicases are indicated by Romannumerals. B, direct amino acid comparison of the six highly conserved helicase regions between NDH II, RNA helicase A (HelA), and Mle protein. Only differences to the NDH II sequence are indicated; dashes represent identical amino acids. Underlined are the NTP binding domains A and B in motif Ia, the DEIH box in motif II, and the conserved nucleic acid binding domain in motif VI.



In addition to the conserved domains, there are further non-canonical helicase motifs present in NDH II. There is a (presumed) nuclear localization sequence, PPPKDKKKD, that is highly conserved between helicase A and NDH II (Fig. 3), while in Mle a comparable lysine-rich motif is found closer toward the N terminus. There are also two putative double-stranded RNA binding domains, named dsRBD I and dsRBD II(26) , at the N-terminal region of NDH II (Fig. 3). Interestingly, the interferon -induced double-stranded RNA-activated protein kinase DAI, which like NDH II has a strong affinity to poly(rIrC), bears similarity to NDH II regarding both dsRBD domains(27) . Therefore, the dsRBD regions might contribute to poly(rIrC) binding.

Most significantly, NDH II contains a glycine-rich region of 121 amino acids at the very C terminus (Fig. 3), comprising 16 imperfect amino acid repeats arranged as GGG(G/D)(Y/V)(G/S/V/R)GG. The glycine-rich domain forms five so called ``RGG'' boxes that are thought to be involved in RNA binding(28, 29, 30) . Similar glycine-rich arrangements have been found in many other proteins that interact with nucleic acids, including ribonuclear particle core proteins A1 (31) and U(28) , the chromatin assembly factor nucleolin(32) , and the Epstein-Barr virus nuclear antigen 1(30) . The multitude of glycine residues may allow to form flexible loop structures; the regularly interspersed amino acids tyrosine/phenylalanine and the positively charged amino acid arginine could provide additional affinity to nucleic acids. Interestingly, the glycine-rich sequence is not stringently conserved within the three Mle proteins. For Drosophila Mle, the numbers of repeats vary between different Mle wild type forms(13) . Although bovine NDH II is strongly homologous to human RNA helicase A, it diverges considerably at the glycine-rich C terminus(11) . Obviously, some deletion mutations must have occurred, which led to different reading frames for the C termini of NDH II and RNA helicase A. Surprisingly, the C-terminal region of bovine NDH II is more similar to the corresponding region of the Drosophila Mle protein than to that of human RNA helicase II.

NDH II Is Encoded by a 4.5-4.7-kb-long mRNA

Both the occurrence of two protein bands in highly purified preparations of NDH II (9) and the observation of untranslatable sequences in several cDNA clones (see ``Experimental Procedures'') suggested that there might have been alternative splicing events at the level of pre-mRNA. Therefore, we analyzed thymus and several other tissues for the presence, the amounts, and sizes of the corresponding mRNAs. Direct hybridization to total RNA of crude cellular extracts gave no positive signals, suggesting that there was only very little NDH II-encoding mRNA present. To increase the signal to noise ratio, we utilized poly(A) RNA from calf thymus as target for Northern blot analysis and a 895-bp-long PCR product obtained from clone 6 as hybridization probe. This probe detected an mRNA transcript migrating at about 4.5-4.7 kb (Fig. 4), a position that was expected from cDNA cloning. With another cDNA probe that contained the glycine-rich region (587 bp from nucleotides 3732 to 4319), again only the 4.5-4.7-kb-long mRNA was detected (data not shown). Visible hybridization signals were only obtained at exposition times of 3 weeks or longer and when more than 2.5 µg of poly(rA) RNA was loaded onto the gel.


Figure 4: Northern blot analysis of poly(A)-containing RNA. Calf thymus poly(A)-containing RNA (2.5 µg) was subjected to the Northern blot analysis, as described under ``Experimental Procedures.'' The size of the RNA for NDH II was estimated to be about 4.5-4.7 kb in length based on its migration to marker RNAs of known length (leftside). Exposure time was 21 days.



NDH II Has a Molecular Mass of 140 kDa in Vivo

In contrast to the low abundant mRNA, NDH II protein could be isolated in milligram amounts from a kilogram of calf thymus. Highly purified preparations of this protein displayed two polypeptides with molecular masses of 130 and 100 kDa(9) . To determine the native form of NDH II in living cells, we undertook a Western blot analysis of calf thymus nuclear extracts with the same anti-NDH II serum used for cDNA screening. Purified NDH II, consisting of p130 and p100, served as control. In nuclear extracts, there was only one protein band with a molecular mass of around 140 kDa (Fig. 5). This molecular weight is in agreement with the molecular weight predicted from the cDNA and strongly suggests that NDH II becomes degraded to p130 and p100 during purification. A similar observation has been made with hnRNP A1(31) : the earlier purified ``unwinding protein 1'' (UP1) (33) was subsequently shown to be a degradation product of hnRNP A1 that arose by deletion of the C-terminal glycine-rich domain(31, 34) .


Figure 5: Western blot analysis of nuclear extracts of calf thymus. Nuclear extract prepared from calf thymus (lanes1 and 3) and purified NDH II (lanes2 and 4) were subjected to the immunoblot analysis using anti-NDH II antibodies (lanes1 and 2) or anti-RNA helicase A antibodies (lanes3 and 4) at a dilution of 1 to 1000 each. The primary antigen-antibody complexes were visualized by an ECL immunodetection procedure using the secondary anti-rabbit IgG antibody at a 1 to 5000 dilution.



Cross-reactivity of Bovine NDH II with Antibodies against Human RNA Helicase A

Since there is a 91.5% amino acid identity among bovine NDH II and human RNA helicase A, we expected a high cross-reactivity with antibodies derived from protein of either species. Antibodies elicited against human RNA helicase A were used to probe the same amount of nuclear extract and purified NDH II on one-half of the membrane shown in Fig. 5. With the same dilution of antibodies, equal intensities of immunostaining were observed as with the antibody directed against the bovine enzyme (Fig. 5). The strong cross-reactivity confirmed the expected homology and, moreover, provided strong evidence for the authenticity of the cDNA clones.

Intracellular Localization of NDH II

To obtain further information on the likely physiological role of NDH II, we examined the intracellular localization of this enzyme by immunofluorescence. For these studies, monkey TC-7 cells were fixed with acetone and subsequently incubated with anti-NDH II antiserum and a secondary, biotinylated anti-rabbit antibody. Incubation with carboxymethylindocyanine-conjugated avidin and subsequent fluorescence microscopy revealed a mainly nuclear localization for NDH II (Fig. 6). NDH II was found at both the nuclear periphery and the nucleoplasm with widespread staining foci throughout the nuclear area. Such a staining pattern does not allow an unambiguous assignment of NDH II to specific locations at either peri- or interchromatin regions. Nevertheless, nucleolar-like structures were not stained, which may allow the conclusion that NDH II is probably not involved in ribosomal RNA synthesis.


Figure 6: Intracellular localization of NDH II. Monkey TC-7 kidney cells were grown to subconfluence on coverslips. After fixation with acetone, the cells were incubated with anti-NDH II serum. Then, the cells were incubated with biotinylated anti-rabbit IgG and subsequently stained with fluorescence dye-conjugated avidin. After addition of mounting solution, the cells were viewed at a 400-fold magnification (A and B) or a 1000-fold magnification (C and D) by using a Zeiss fluorescence microscope and the fluorescein Cy3 excitation wavelength. The staining of the cytosol and the perinuclear region was also visible with control serum (B and D).



Comparison of Bovine NDH II and Human RNA Helicase A

The extensive sequence homology between bovine NDH II and its human homologue RNA helicase A is reflected by very similar biochemical properties, such as a comparable molecular weight, the same directionality of unwinding, and the capability for using all four dNTPs and rNTPs(10, 12) . RNA helicase A binds to DNA as demonstrated by affinity chromatography on single-stranded DNA cellulose; furthermore, its ATPase can be stimulated by poly(dT) and poly(dI) (12). Despite this and in contrast to NDH II, RNA helicase A is apparently unable to unwind DNA(12) . However, as we have shown earlier, NDH II-catalyzed DNA unwinding is salt sensitive, leading to a complete inhibition of the DNA unwinding activity in the presence of 75 mM NaCl(9) . On the other hand, RNA unwinding is barely affected by 75 mM salt(10) . Therefore, we suspect that a DNA unwinding capability of RNA helicase A has remained undetected because of too high salt conditions in the corresponding assay.

Possible Physiological Role(s) of Mle Proteins

The demonstration of a DNA helicase activity for NDH II might provide a novel clue for the function of the highly homologous and genetically characterized Mle protein from fruit flies. Mle helicase is required for the equalization of the X-linked gene dosage between the two sexes, probably by altering the chromatin structure of the X chromosome to increase transcription from this chromosome(35) . The 85% similarity between Mle and NDH II speaks in favor of a conserved function for both proteins. However, sex determination in mammals is completely different from that in Drosophila. In mammals, the only X chromosome of males contributes as much as both X chromosomes of females, because one of the female X chromosomes is randomly inactivated. Hence, we cannot expect that NDH II performs a role comparable to that of Mle protein in Drosophila. But there is also evidence for Mle being involved in non-sex-specific processes. For example, the Mle mutation nap affects in both sexes the expression of the gene para, which encodes a sodium channel essential for the membrane excitability in the initiation and propagation of the action potential(36, 37) . NAP contains an amino acid substitution near the nucleotide binding motif of Mle, which in turn may lead to an impaired helicase function and consequently an inefficient expression of the sodium channel(37) . A still hypothesized DNA unwinding function of Mle protein (analogous to that of NDH II) might help to decondense the chromatin structure; alternatively, DNA unwinding might increase RNA polymerase II-dependent transcription by either enhancing strand opening during elongation or facilitating the release of the transcript. Further experiments are necessary to distinguish between these alternatives and to assign physiological function(s) to this class of highly conserved helicases.


FOOTNOTES

*
This work was supported by Grant Gr 895/5-2 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X82829.

§
Present address: Institut für Molekulare Biotechnologie, Abteilung Biochemie, Postfach 100 813, D-07708 Jena, Germany.

To whom correspondence should be addressed. Tel.: 49-3641-656285; Fax: 49-3641-656288.

The abbreviations used are: NDH, nuclear DNA helicase; PCR, polymerase chain reaction; PBS phosphate-buffered saline; Cy3, carboxymethylindocyanine-conjugated avidin; bp, base pair(s); kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Jerard Hurwitz (Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York) for the kind gift of anti-human RNA helicase A antibodies. We also thank Torsten Mummenbrauer (The Heinrich-Pette-Institut (HPI, Hamburg), which is financially supported by the Freie und Hansestadt Hamburg and Bundesministerium für Gesundheit) for help with the computer-assisted sequence comparisons, and Wolfgang Deppert (HPI) for critically reading the manuscript.


REFERENCES
  1. Kornberg, A., and Baker, T. (1991) in DNA Replication, 2nd Ed., pp. 373-378, W. H. Freeman, San Francisco, CA
  2. Matson, S. W., and Kaiser-Rogers, K. A. (1990) Annu. Rev. Biochem.59, 289-329 [CrossRef][Medline] [Order article via Infotrieve]
  3. Matson, S. W. (1991) Progr. Nucleic Acids Res. Mol. Biol.40, 289-327 [Medline] [Order article via Infotrieve]
  4. Thömmes, P., and Hübscher, U. (1990) FEBS Lett.268, 325-328 [CrossRef]
  5. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P., and Blinov, V., M. (1989) Nucleic Acids Res.17, 4713-4730 [Abstract]
  6. Bork, P., and Koonin, E. V. (1993) Nucleic Acids Res.21, 751-752 [Medline] [Order article via Infotrieve]
  7. Linder, P., Lasko, P. F., Ashburner, M., Leroy, P., Nielsen, P. J., Nishi, K., Schnier, J., and Slonimski, P. P. (1989) Nature337, 121-122 [CrossRef][Medline] [Order article via Infotrieve]
  8. Wassarman, D. A., and Steitz, J. A. (1991) Nature349, 463-464 [CrossRef][Medline] [Order article via Infotrieve]
  9. Zhang, S., and Grosse, F. (1991) J. Biol. Chem.266, 20483-20490 [Abstract/Free Full Text]
  10. Zhang, S., and Grosse, F. (1994) Biochemistry33, 3906-3912 [Medline] [Order article via Infotrieve]
  11. Lee, C.-G., and Hurwitz, J. (1993) J. Biol. Chem.268, 16822-16830 [Abstract/Free Full Text]
  12. Lee, C.-G., and Hurwitz, J. (1992) J. Biol. Chem.267, 4398-4407 [Abstract/Free Full Text]
  13. Kuroda, M. I., Kernan, M. J., Kreber, R., Ganetzky, B., and Baker, B. S. (1991) Cell66, 935-947 [Medline] [Order article via Infotrieve]
  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY
  15. Ochman, H., Medhora, M. M., Garza, D., and Harte, D. L. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 219-227, Academic Press, San Diego, CA
  16. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Science243, 217-220 [Medline] [Order article via Infotrieve]
  17. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem.162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  18. Grosse, F., and Manns, A. (1993) Methods in Molecular Biology: Enzymes of Molecular Biology (Burrell, M. M., ed) pp. 95-105, Humana Press, Totowa, NJ
  19. Laemmli, U. K. (1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  20. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J.1, 945-951 [Medline] [Order article via Infotrieve]
  21. Shuman, S. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 10935-10939 [Abstract]
  22. Pause, A., Methot, N., and Sonenberg, N. (1993) Mol. Cell. Biol.13, 6789-6798 [Abstract]
  23. Chen, J. H., and Lin, J. (1990) Nucleic Acids Res.18, 6447 [Medline] [Order article via Infotrieve]
  24. Strauss, E. J., and Guthrie, C. (1991) Genes & Dev.5, 629-641
  25. Company, M., Arenas, J., and Abelson, J. (1991) Nature349, 487-493 [CrossRef][Medline] [Order article via Infotrieve]
  26. Gibson, T. J., and Thompson, J. D. (1994) Nucleic Acids Res.22, 2552-2556 [Abstract]
  27. Johnston, D. S., Brown, N. H., Gall, J. G., and Jantsch, M. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 10979-10983 [Abstract]
  28. Kiledjian, M., and Dreyfuss, G. (1992) EMBO J.11, 2655-2664 [Abstract]
  29. Dreyfuss, G., Matunis, M. J., Piol-Roma, S., and Burd, C. G. (1993) Annu. Rev. Biochem.62, 289-321 [CrossRef][Medline] [Order article via Infotrieve]
  30. Snudden, D. K., Hearing, J., Smith, P. R., Grässer, F. A., and Griffin, B. E. (1994) EMBO J.13, 4840-4847 [Abstract]
  31. Cobianchi, F., SenGupta, D. N., Zmudzka, B. Z., and Wilson, S. H. (1986) J. Biol. Chem.261, 3536-3543 [Abstract/Free Full Text]
  32. Ghisolfi, L., Joseph, G., Amalric, F., and Erard, M. (1992) J. Biol. Chem.267, 2955-2959 [Abstract/Free Full Text]
  33. Herrick, G., and Alberts, B. (1976) J. Biol. Chem.251, 2124-2132 [Abstract]
  34. Riva, S., Morandi, C., Tsoulfas, P., Pandolfo, M., Biamonti, G., Merrill, B., Williams, K. R., Multhaupt, G., Beyreuther, K., Werr, H., Henrich, B., and Schäfer, K. P. (1986) EMBO J.5, 2267-2273 [Abstract]
  35. Gorman, M., and Baker, B. S. (1994) Trends Genet.10, 376-380 [CrossRef][Medline] [Order article via Infotrieve]
  36. Loughney, K., Kreber, R., and Ganetzky, B. (1989) Cell58, 1143-1154 [Medline] [Order article via Infotrieve]
  37. Kernan, M. J., Kuroda, M. I., Kreber, R., Baker, B. S., and Ganetzky, B. (1991) Cell66, 949-959 [Medline] [Order article via Infotrieve]
  38. Marck, C. (1988) Nucleic Acids Res.16, 1829-1836 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.