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A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.

In an attempt to identify chlamydial genes whose native promoters allow them to be expressed in Escherichia coli, we isolated and characterized a chlamydial gene identified by screening a library of chlamydial DNA with antichlamydial antibodies. This gene encodes a 70-kilodalton immunoreactive polypeptide in E. coli hosts. Sequence analysis of the 5' portion of the gene identified its product as the chlamydial homolog of the E. coli ribosomal protein S1. The site of transcription initiation of the mRNA in chlamydiae was determined, and its putative promoter regions were identified. These regions apparently do not function efficiently in E. coli; in vitro transcripts generated by using E. coli RNA polymerase did not start at the authentic chlamydial initiation site. Several in vitro transcripts both larger and smaller than the authentic transcript were seen; presumably, these transcripts result from adventitious promoterlike elements in adjacent chlamydial DNA and may be responsible for the expression of the gene in E. coli.

To determine which of the major ground squirrel hepatitis virus RNAs serve as mRNAs and which serve as templates for reverse transcription of the genome, we analyzed the subcellular distribution of these RNAs in livers of infected ground squirrels. Both major classes of viral RNA, the 2.3- and 3.5-kilobase (kb) classes, are unspliced, are polyadenylated at a common position, and display heterogeneous 5' ends that can encode proteins with different amino termini (G.H. Enders, D. Ganem, and H. Varmus, Cell 42:297-308, 1985). Both of the 2.3-kb RNAs, which encode surface antigens, appear to be predominantly associated with polyribosomes. Of the three 3.5-kb RNAs, the two longer, which can encode a protein initiated from the first methionine codon in the core antigen gene, appear to be predominantly associated with polyribosomes, and a minority of the shortest 3.5-kb RNAs, which can encode a protein initiated from the second methionine in the core antigen gene, appears to be associated with polyribosomes. This last RNA is instead found predominantly within viral core particles, consistent with evidence that indirectly implicates it in two steps of viral DNA synthesis (C. Seeger, D. Ganem, and H.E. Varmus, Science 232:477-484, 1986). None of the other viral RNAs is detectably packaged into cores. These findings provide independent evidence that the shortest 3.5-kb RNA is the template for synthesis of the viral genome and reveal a novel selectivity in viral RNA packaging.

We isolated and characterized the rRNA operons of murine Chlamydia trachomatis. By exhaustively screening a library of chlamydial DNA and by blot hybridization of genomic DNA, we showed that there are only two rRNA operons in C. trachomatis. S1 nuclease protection and primer extension analysis were used to map the 5' and 3' ends of the mature 16S and 23S transcripts in both rRNA cistrons and, additionally, to demonstrate the lack of intervening sequences in these genes. The 5' ends of the presumed primary rRNA transcript were located and found to originate at two tandem sites separated by 100 base pairs. The two tandem chlamydial rDNA transcripts were not differentially regulated. Their products were coordinately expressed and were detectable as early as 9 h postinfection. However, the upstream transcript was only 10% as abundant as the downstream transcript. The sequences surrounding the transcription initiation sites bore little homology with each other or with the classic Escherichia coli -10 and -35 promoter sequences. This finding suggests that chlamydial transcription signals may differ from those of previously studied procaryotes.

The coding region for the major polypeptide (p24S) of hepatitis B surface antigen (HBsAg) is preceded by an in-phase open reading frame termed pre-S. The coding potential of the pre-S region was examined in mouse L cells transformed with cloned hepatitis B virus DNA. Such cells produce three HBsAg-related polypeptides of Mr 24,000, 27,000, and 35,000 organized into complex particles of 22 nm diameter. These HBsAg particles bind to polymerized human albumin, but not to polyalbumins of several other species. In contrast, cells transformed with hepatitis B virus DNA bearing a frameshift mutation near the 3' end of the pre-S region secrete immunoreactive HBsAg particles containing only the 24,000 and 27,000 Mr species. These mutant particles, which lack the 35,000 Mr species, are unable to bind polymerized human albumin. These studies indicate that the pre-S region encodes the 35,000 Mr species, that this product accounts for the known polyalbumin-binding activity of HBsAg but is not required for assembly and secretion of HBsAg 22-nm particles, and that the major polypeptide of HBsAg is not derived primarily by cleavage of larger precursors encoded by the pre-S region.

Hepatitis B viruses synthesize their open circular DNA genomes by reverse transcription of an RNA intermediate. The details of this process have been examined with the use of mammalian hepatitis B viruses to map the sites for initiation and termination of DNA synthesis and to explore the consequences of mutations introduced at short, separated direct repeats (DR1 and DR2) implicated in the mechanisms of initiation. The first DNA strand to be synthesized is initiated within DR1, apparently by a protein primer, and the completed strand has a short terminal redundancy. In contrast, the second DNA strand begins with the sequence adjacent to DR2, but its 5' end is joined to an oligoribonucleotide that contains DR1; thus the putative RNA primer has been transposed to the position of DR2. It is now possible to propose a detailed strategy for reverse transcription by hepatitis B viruses that can be instructively compared with that used by retroviruses.

Hepatitis B viruses of humans, woodchucks, ground squirrels, and ducks are similar biochemically but differ with respect to host range and pathogenicity. To pursue the genetic basis of these properties in the absence of a cell culture system for virus growth, we exploited the demonstrated infectivity of cloned hepatitis B virus DNA in whole animals. We constructed several recombinant molecules in vitro between cloned infectious genomes of woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV) and assayed the recombinants for infectivity after intrahepatic injection in ground squirrels, which support growth of GSHV but not WHV. Two of the recombinants molecules initiated productive infection; in one recombinant genome, 76% of the coding region for the major surface glycoprotein of GSHV and for the overlapping portion of the presumptive gene for DNA polymerase was replaced by WHV DNA; in the other, 29% of the same coding domain was replaced by WHV DNA. These findings demonstrate the feasibility of generating viable recombinants of hepatitis B viruses from different animal species and suggest that the major host range determinants are not encoded within the surface antigen gene of these viruses.

The ground squirrel hepatitis virus (GSHV) belongs to a recently defined class of viruses with open circular DNA genomes that encode proteins in extensively overlapping reading frames and appear to replicate via RNA intermediates. We have determined the primary structure of the major GSHV transcripts in the livers of infected ground squirrels. Both major classes of transcripts, 2.3 kb and 3.5 kb, are plus-stranded, unspliced, polyadenylated at a common position, and display heterogeneous 5' ends that can encode proteins with different amino termini. The 2.3 kb transcripts, like their structural analogs transcribed from human hepatitis B virus DNA, are likely mRNAs for products of the major surface antigen and presurface coding domains. The 3.5 kb transcripts are likely mRNAs for one or more products of the core antigen reading frame; these transcripts also encompass the entire genome and contain terminal redundancies of 130-160 nucleotides that include a putative initiation site for reverse transcription.

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.

To investigate the mechanism by which complex membrane proteins achieve their correct transmembrane orientation, we examined in detail the hepatitis B surface antigen for sequences which determine its membrane topology. The results demonstrated the presence of at least two kinds of topogenic elements: an N-terminal uncleaved signal sequence and an internal element containing both signal and stop-transfer function. Fusion of reporter groups to either end of the protein suggested that both termini are translocated across the membrane bilayer. We propose that this topology is generated by the conjoint action of both elements and involves a specifically oriented membrane insertion event mediated by the internal sequence. The functional properties of each element can be instructively compared with those of simpler membrane proteins and may provide insight into the generation of other complex protein topologies.

Retroviruses and many other types of genetic elements replicate by reverse transcription of RNA. Although structurally and biologically very diverse, such elements carry conserved polymerase genes (pol) that encode proteins required for reverse transcription. In most cases, the pol gene is preceded by an overlapping gene encoding one or more nucleocapsid proteins, in a different reading frame. Because both coding regions are represented in a single mRNA, the question arises of how the reverse transcriptase in the alternative reading frame is expressed. In retroviruses and retrotransposons it is expressed as a nucleocapsid-polymerase fusion protein by ribosomal frameshifting during translation of the overlapping region. We have examined the mechanism of polymerase biosynthesis in another family of animal viruses that use reverse transcription, the hepatitis B viruses. Genetic and biochemical studies reveal that these viruses do not use ribosomal frameshifting to generate this enzyme, but instead direct translation initiation at an internal initiation (AUG) codon in the polymerase gene.

All hepadnaviruses produce multiple genome-length RNA species, only one of which is encapsidated into subviral core particles prior to reverse transcription. To study the encapsidation mechanism, we developed a system in which the packaging of genetically marked target genomes of duck hepatitis B virus is mediated by factors supplied from a separate (helper) plasmid that encodes encapsidation functions. In the helper plasmid, the synthesis of the viral core and polymerase proteins was driven by a simian virus 40 promoter; the RNA produced by this construct was itself inefficiently packaged and was not active as a template for reverse transcription. Cotransfection of this construct with mutant genomes bearing frameshift lesions in either core or polymerase cistrons resulted in the successful packaging and reverse transcription of the mutant genomes. This system should allow definition of both the cis- and trans-acting elements of the encapsidation pathway.

Cloned hepatitis B virus (HBV) DNA directs the synthesis of the viral surface antigen (HBsAg) when introduced into mouse L cells by DNA transformation. We have used recombinants between the Rous sarcoma virus long terminal repeat and subgenomic fragments of HBV DNA to localize regions of the HBV genome required for HBsAg expression. Examination of HBV-specific RNA from such transformants indicates that transcription initiates at three distinct sites (153, 163, and 183 nucleotides upstream from the translation initiation codon for mature HBsAg). Thus in these cells, a large segment of the presurface reading frame is not represented in HBsAg mRNA. The termination site of this RNA lies within the coding sequences for the viral core antigen, some 1,094 +/- 10 base pairs downstream from the TAA stop codon for HBsAg. Two additional open reading frames are present in the resultant unspliced HBsAg RNA.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.

The preS1 surface glycoprotein of hepatitis B virus is targeted to the endoplasmic reticulum (ER) and is retained in this organelle when expressed in the absence of other viral gene products. The protein is also acylated at its N terminus with myristic acid. Sequences responsible for its ER retention have been identified through examination of mutants bearing lesions in the preS1 coding region. These studies reveal that such sequences map to the N terminus of the molecule, between residues 6 and 19. Molecules in which this region was present remained in the ER; those in which it had been deleted were secreted from the cell. Although all deletions which allowed efficient secretion also impaired acylation of the polypeptide, myristylation alone was not sufficient for ER retention: point mutations which eliminated myristylation did not lead to secretion. These data indicate that an essential element for ER retention resides in a 14-amino-acid sequence that is unrelated to previously described ER retention signals.

The developmental cycle of the obligate intracellular prokaryote Chlamydia trachomatis involves the serial alternation of two distinct morphological forms of the organism. To examine the basis of chlamydial differentiation we have searched for developmentally regulated gene products in this species. Chlamydia-infected cells were pulse-labelled with [35S] cysteine at various stages of development and the products of synthesis examined by SDS-PAGE. Our results indicate that the synthesis of the cysteine-rich outer-membrane proteins is developmentally regulated, occurring only late in the cycle during the conversion of reticulate bodies to elementary bodies. Both hydroxyurea and ampicillin block this conversion; as a result of this blockade the cysteine-rich outer-membrane proteins are not produced in the presence of either drug.

To identify possible intermediates in the replication of ground squirrel hepatitis virus, we characterized the major forms of intracellular virus-specific DNA in the livers of infected ground squirrels. A variety of DNA species were found: covalently closed circular molecules, relaxed circular molecules, and a heterogeneous collection of molecules that migrated ahead of closed circular DNA during agarose gel electrophoresis. The heterogeneous DNA was at least partly single stranded, consisted of minus strands in a greater than eight-fold mass excess of plus strands, and was tightly associated with protein.

The cloned DNA of duck hepatitis B virus (DHBV) can initiate a productive infection of susceptible ducklings when presented by direct intrahepatic injection. We have examined the effects of the structure of the incoming DNA upon the outcome of this in vivo transfection. Plasmid-linked DHBV genomes of greater than unit length regularly give rise to infectious virus irrespective of the location of the plasmid insert on the transcriptional map. When mutant DHBV genomes are coinjected with subgenomic viral DNA fragments spanning the mutation, wild-type recombinants arise. These results indicate that, as in transfection of cultured cells, in vivo transfection of hepatocytes is regularly followed by homologous recombination involving incoming DNA molecules. These frequent recombination events will complicate efforts to use in vivo transfection for certain genetic analyses of hepadnaviruses.

Although most infections of humans with hepatitis B virus are self-limited, up to 10% of individuals experiencing primary infection remain persistently infected. These individuals are usually asymptomatic but may suffer episodic or progressive liver injury that can result in cirrhosis and liver failure. In addition, chronic infection with hepatitis B virus is, in certain settings, associated with the late development of primary hepatocellular carcinoma. This review summarizes the biological characteristics of persistent viral infection, with particular emphasis on host and viral factors that affect the outcome of primary infection.

Cloned DNA of duck hepatitis B virus (DHBV) was used to transfect two differentiated human hepatoma cell lines, Huh 7 and Hep G2. Use of the transfected genome as a transcriptional template was demonstrated by the appearance of virus-specific subgenomic and genomic transcripts. Comparison of the steady-state ratio of subgenomic to genomic transcripts in Huh 7 and Hep G2 cells suggests that there are differences in the relative stability and/or rate of production of these transcripts between these cell lines. Viral genomic replication proceeded in both lines, as judged by the presence of DHBV DNA replicative intermediates in cytoplasmic core particles; the levels of these replicative intermediates is roughly equivalent in Huh 7 and Hep G2 cells. Subcutaneous injection of tissue culture medium from transfected Huh 7 cells into Pekin ducks resulted in productive DHBV infection, indicating the production and export of biologically active virus. These cell lines should provide a valuable system for studying the molecular mechanisms of the hepadnaviral life cycle.

The lack of an in vitro infectivity assay for hepatitis B viruses has impeded the analysis of their genetic organization. To examine the feasibility of generating mutant and recombinant viruses after manipulation of cloned viral DNA in vitro, we have tested the infectivity of the cloned genome of ground squirrel hepatitis virus (GSHV) in virus-free Beechey ground squirrels. We demonstrate that cloned GSHV DNA is infectious when injected directly into the liver in the form of trimeric, head-to-tail recombinant clones and recircularized monomeric molecules but not when injected into the portal vein. Infections established in all four recipients of intrahepatic injections of cloned GSHV DNA exhibited the characteristics observed after administration of virus: GSHV surface antigen and viral DNA appeared in the serum 14-22 weeks after inoculation, and both circular and heterogeneous protein-linked forms of viral DNA were found in liver biopsy samples. Furthermore, virus present in the sera of these animals can be transmitted to other ground squirrels. These findings imply that any function of virion proteins in the initiation of infection by hepatitis B viruses can be bypassed with the use of cloned viral DNA and that this animal model is suitable for testing mutant genomes.

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also independently secreted from infected cells as a lipoprotein particle. Secretion proceeds without signal sequence removal or cleavage of other segments of the polypeptide. We have examined the synthesis and transport of HBsAg in cultured cells expressing the cloned surface antigen gene. Our results show that HBsAg is initially synthesized as a integral membrane protein. This transmembrane form is slowly converted to a secreted lipoprotein complex in the lumen of the endoplasmic reticulum via a series of definable intermediates, after which it is secreted from the cell. This unusual export process shares many features with the assembly and budding reactions of conventional enveloped animal viruses. However, it differs importantly in its absence of a requirement for the participation of nucleocapsid or other viral proteins.

Ground squirrel hepatitis virus (GSHV) is a small DNA virus, structurally and antigenically related to the human hepatitis B virus, which occurs naturally among certain wild populations of ground squirrels (P. L. Marion et al., Proc. Natl. Acad. Sci. U.S.A. 77:2941-2945, 1980). Serum from naturally infected animals was used to transmit GSHV in the laboratory by parenteral inoculation of susceptible squirrels. Sixty percent of recipient animals developed viral surface antigenemia after a latent period of 2 to 3 months; three of these animals have remained viremic for over 9 months. Like hepatitis B virus, GSHV demonstrates marked hepatotropism, with viral DNA detected in significant quantities only in the liver, where an average of 6 X 10(2) to 6 X 10(3) viral DNA molecules per cell were found by molecular hybridization. However, histological signs of liver injury after acute infection are minimal. In contrast to infection of its natural host, parenteral administration of GSHV to rats, mice, guinea pigs, and hamsters did not result in demonstrable antigenemia, suggesting that the host range of GSHV, like that of hepatitis B virus, is narrow.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.

By the use of a truncated recombinant hepatitis B virus polymerase antigen, we have characterized a series of patient sera for anti-hepatitis B virus polymerase antibodies. Seven of 54 (13%) had antipolymerase antibodies detectable by Western blot analysis, and no close correlation was apparent between the disease status and patient's immune response against hepatitis B virus polymerase antigen. Our results indicate that serologic responses to the viral polymerase are demonstrable but suggest that such antibodies are not likely to be clinically useful as diagnostic or prognostic markers of infection.

The infectivity and host range of ground squirrel hepatitis virus (GSHV) have been further examined by animal inoculation experiments. Although carrier squirrel sera usually harbor 10(9) to 10(10) virions per ml as determined by physical measurements, titration of one such serum revealed that squirrel infectivity was lost following dilution of the sample over 10(6)-fold. Infectivity is markedly reduced by NP40 pretreatment of infected serum. GSHV infection cannot be readily transmitted to several related ground squirrel species, but chipmunks can be experimentally infected by GSHV virions or by cloned GSHV DNA, and the resulting infection closely resembles that seen in the normal host.

Hepatitis B surface antigen is the major protein of the virion envelope, and is also independently secreted from infected cells as a subviral particle composed exclusively of HBsAg and host-derived lipid. Similar particles are efficiently assembled and secreted by cultured mammalian cells transfected with the gene for HBsAg. In contrast to such cultured cells, Xenopus oocytes microinjected with HBsAg mRNA secrete less than 5% of newly synthesized HBsAg polypeptides. We have examined the HBsAg biosynthetic intermediates in such oocytes and provide evidence that the impaired secretion of HBsAg is due to a discrete block in the assembly of lipoprotein particles.

A technique is described for mapping point mutations in the first 59 amino-acid residues of the lac repressor from Escherichia coli, using less than 0.1 mumol (4 mg) of the purified protein. This technique was used to localize five mutations affecting the ability of the i-gene product to repress in vivo. These alterations are located at four different sites in the amino-terminal region of the repressor molecule. Three of these are missense mutations and result in changes from serine to proline (residue 16), threonine to alanine (residue 19), and alanine to valine (residue 53). Each amino-acid substitution alone is sufficient to eliminate repression in vivo, presumably by altering the operator binding activity. The remaining two independently-isolated mutations are identical, and result in a change from a glutamine codon at position 26 to an amber (UAG) codon. Since suppression of this nonsense mutation with amber suppressors that insert leucine, tyrosine, serine, or glutamine restores repressor activity to the molecule, glutamine(26) cannot be crucial for the operator-binding function. A comparison of the position of each altered residue with the genetic map enabled us to estimate the physical distance between several deletion-group endpoints.

52 Spontaneous nonsense mutants in the lac i gene of Escherichia coli were isolated and characterized. All mutants located early in the gene show negative complementation in vivo with a wild-type i gene in a recA diploid strain. In vitro studies show that those mutants that display negative complementing activity in vivo also make lac repressor fragments retaining inducer binding and immunological crossreactivity with wild-type repressor. Amino-acid sequence analysis of these fragments shows that they arise by reinitiation at internal sities of the i message after chain termination at a prior amber or ochre codon. There are at least two different internal reinitiation sites in the first 200 nucleotides of the translated part of the i message. The first site corresponds to the first internal in phase AUG codon, which specifies the methionine residue at position 42 of the repressor protein. This site can be activated by an amber codon, 45 nucleotides before the AUG codon. The second site is only 60 nucleotides past the first site and can be activated by an amber mutation derived from residue 60 of the protein. The second initiation codon specifies the amino-acid leucine in the wild-type repressor, but the reinitiated fragment shows an amino-terminal methionine residue at this position. Therefore, the second initiation site seems to involve an in vivo ambiguity of the genetic code in that the same codon can be translated into two different amino acids depending on the recognition of this codon during initiation (when methionine is inserted) or elongation of protein synthesis (when leucine is inserted). The possibility that a codon other than AUG can act as an initiation codon in vivo is discussed.

An early, spontaneous amber mutation in the lac i-gene allows translational reinitiation, which results in a mutant lac repressor. Comparison of the amino-terminal sequence of this mutant repressor with the partial amino-acid sequence of the wild-type lac repressor shows that reinitiation occurs at the first internal AUG codon, and results in a mutant protein lacking 42 residues at the amino-terminal end. This protein binds the inducer isopropyl-beta-D-thiogalactoside with normal affinity, and is capable of maintaining a tetrameric structure; however, it does not repress in vivo. These data suggest that the amino-terminal portion of the wild-type lac repressor is necessary either for direct binding to the lac operator or for the correct conformation for binding to DNA.