Slp Gene Research Articles

Evaluating The Cytotoxic Activity of Lactobacillus plantarum IIA-1A5 Against MCF-7 Human Breast Cancer Cells and Identifying Its Surface Layer Protein Gene

Breast cancer is a serious global health concern, with a high mortality rate worldwide. Using natural compounds as potential cancer therapies is a promising approach to address this issue. Previous research has shown that probiotic lactic acid bacteria (LAB) and their metabolites, such as surface layer protein (slp), have a positive impact on a variety of health disorders, including cancer. The purpose of this study was to evaluate the ability of Lactobacillus plantarum IIA-1A5 to suppress the growth of the MCF-7 breast cancer cell line and detect the presence of the slp gene. Intracellular and extracellular protein fractions were isolated from L. plantarum IIA-1A5 cultures. The protein concentrations and molecular weights of the extracts were measured. The anticancer activity of the extracts was assessed using the MTT cytotoxicity test, and IC50 values were calculated. The slp gene was identified through polymerase chain reaction (PCR) amplification and nucleotide sequencing. The results demonstrated that L. plantarum IIA-1A5 had a concentration-dependent inhibitory effect on MCF-7 breast cancer cells, with IC50 values of 6.831 and 12.35 μg/mL for intracellular extracts and extracellular extracts, respectively. Additionally, PCR amplification and nucleotide sequencing confirmed the presence of the slp gene, which may contribute to the strain’s anticancer abilities. These findings suggest the potential of L. plantarum IIA-1A5 as a natural anticancer agent against MCF-7 breast cancer cells. Further research is warranted to elucidate the underlying mechanisms of L. plantarum IIA-1A5 in breast cancer treatment.

The Escherichia coli O157:H7 carbon starvation-inducible lipoprotein Slp contributes to initial adherence in vitro via the human polymeric immunoglobulin receptor.

Escherichia coli O157:H7 is the most well-studied serotype of the enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 bacteria and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of the slp gene fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested and not the nonpathogenic control strain E. coli K12. Additionally, deletion of slp gene resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp gene complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.

LC-MS/MS Analysis of Surface Layer Proteins as a Useful Method for the Identification of Lactobacilli from the Lactobacillus acidophilus Group

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/ MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

Assessment of bile salt effects on s-layer production, slp gene expression and some physicochemical properties of Lactobacillus acidophilus ATCC 4356.

In many conditions, bacterial surface properties are changed as a result of variation in growth medium and conditions. This study examined the influence of bile salt concentrations (0-0.1%) on colony morphotype, hydrophobicity, H2O2 concentration, S-layer protein production and slpA gene expression in Lactobacillus acidophilus ATCC 4356. It was observed that two types of colonies (R and S) were in the control group and the stress condition. When the bile level increased in the medium, the amount of S type was more than the R. A stepwise increment in the bile concentration resulted in a stepwise decline in the maximum growth rate. The results showed that hydrophobicity was increased in 0.01%-0.02% bile but it was decreased in 0.1% bile. Treatment by bile (0.01%- 0.1%) profoundly decreased H2O2 formation. S-layer protein and slpA gene expression was also altered by stress condition. S-protein expression was increased in stress condition. slpA gene expression increased in 0.01%-0.05% bile and it decreased in 0.1% bile. However, we found that different of bile salt concentrations influence on morphology and some surface properties of L. acidophilus ATCC 4356. These changes were very different in the 0.1% bile. It appears that the bacteria respond abruptly to 0.1% bile.

Characterization, Identification, and Cloning of the S-Layer Protein from Cytophaga sp.

We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29-48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression.

Robustness and modular design of the Drosophila segment polarity network

Biomolecular networks have to perform their functions robustly. A robust function may have preferences in the topological structures of the underlying network. We carried out an exhaustive computational analysis on network topologies in relation to a patterning function in Drosophila embryogenesis. We found that whereas the vast majority of topologies can either not perform the required function or only do so very fragilely, a small fraction of topologies emerges as particularly robust for the function. The topology adopted by Drosophila, that of the segment polarity network, is a top ranking one among all topologies with no direct autoregulation. Furthermore, we found that all robust topologies are modular—each being a combination of three kinds of modules. These modules can be traced back to three subfunctions of the patterning function, and their combinations provide a combinatorial variability for the robust topologies. Our results suggest that the requirement of functional robustness drastically reduces the choices of viable topology to a limited set of modular combinations among which nature optimizes its choice under evolutionary and other biological constraints.

Regulatory capacity of an androgen-specific enhancer of the mouse Slp gene in transgenic mice

Different steroid hormone receptors can activate transcription from the same hormone response element (HRE) in vitro, but in vivo the effects of each hormone on gene activity are distinct. To determine sequences mediating androgen-specific response in a physiological setting, we placed the androgen-responsive mouse sex-limited protein gene ( Slp) enhancer before a tkCAT reporter in transgenic mice. The enhancer contains a consensus HRE plus accessory factor binding sites that act in concert to direct transcription in response to androgen. A 160 bp fragment, C′Δ2, is responsive to several steroids in transfection; in transgenic mice, this enhancer was active in several tissues of male and female mice, in four of six transgenic lines. In striking contrast, C′Δ9, a 120 bp sub-fragment of C′Δ2 that responds only to androgen in transfection, showed activity in testes, prostate and kidney, where it was strongly androgen-inducible in females. However, expression was obtained in only one transgenic line. Multimerization of the C′Δ9 enhancer conferred expression in prostate, but again in only one line. The greater penetrance of C′Δ2 expression was not driven by glucocorticoids, as adrenalectomy had little effect, but may be dependent on the NF- κB-like element absent from the C′Δ9 fragment. That two transgenic lines showed expression in androgen target sites driven by enhancers that are androgen-specific in vitro suggested that activation of this enhancer, when it could occur, was in response to androgen. The dramatically different behavior of the two related enhancer sequences underscores the importance of chromosomal context to the activity and specificity of regulatory elements.

Involvement of an octamer-like sequence within a crucial region of the androgen-dependent Slp enhancer.

Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, both in vitro and in vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased Oct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C' delta9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C 'delta2). This underscores that, despite extensive sequence identity of C' delta9 and C' delta2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid responsive.

Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver.

Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.

Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments.

During germ-band extension in the Drosophila embryo, intercellular communication is required to maintain gene expression patterns initiated at cellular blastoderm. For example, the wingless (wg) single-cell-wide stripe in each parasegment (PS) is dependent on a signal from the adjacent, posterior cells, which express engrailed (eN). This signal is thought to be the hedgehog (hh) gene product, which antagonizes the activity of patched (ptc), a repressor of wg expression. Genetic evidence indicates that the hh signal is bidirectional, but wg transcription is only derepressed on the anterior side of the en/hh stripes. To explain the asymmetric response of the wg promoter to the hh signal, current models predict that each PS is divided into cells that are competent to express either wg or en, but not both. The sloppy paired (slp) locus contains two transcription units, both encoding proteins containing a forkhead domain, a DNA-binding motif. Removal of slp gene function causes embryos to exhibit a severe pair-rule/segment polarity phenotype. We show that the en stripes expand anteriorly in slp mutant embryos and that slp activity is an absolute requirement for maintenance of wg expression at the same time that wg transcription is dependent on hh. The slp proteins are expressed in broad stripes just anterior of the en-positive cells, overlapping the narrow wg stripes. We propose that by virtue of their ability to activate wg and repress en expression, the distribution of the slp proteins define the wg-competent and en-competent groups. Consistent with this hypothesis, ubiquitous expression of slp protein throughout the PS abolishes en expression and, in ptc mutant embryos, results in a near ubiquitous distribution of wg transcripts. In addition to demonstrating the role of slp in maintaining segment polarity, our results suggest that slp works in, or parallel with, the ptc/hh signal transduction pathway to regulate wg transcription.

Characterization of the carbon starvation-inducible and stationary phase-inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli.

Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide-deficient phoA reporter sequence to a csi (carbon starvation-inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild-type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locus slp (starvation lipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. The slp gene was located at 78.6 centisomes on the E. coli genetic map. The -10 and -35 regions upstream of the mRNA start site were characteristic of a sigma 70 promoter. The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame. Induction of beta-galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation-inducible or stationary phase-inducible proteins.

An element regulating adrenal-specific steroid 21-hydroxylase expression is located within the slp gene.

In this report we demonstrate that a transcriptional regulatory element for one gene lies within a second, seemingly unrelated gene. Specifically, the 3' portion of the murine sex-limited protein (slp) gene, located within the class III region of the major histocompatibility complex, contains an element that regulates expression of the linked steroid 21-hydroxylase gene. A 4.2-kilobase (kb) major histocompatibility complex region, located between -2.2 and -6.4 kb upstream of 21OH-A, is required for expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Two short regions of DNA, located between -5.3 and -6.0 kb, stimulate chloramphenicol acetyltransferase expression in Y1 adrenocortical tumor cells, and both of these active regions lie within the slp gene. A 21-base pair sequence, which is required for activity of the most 3' region, does not contain any of over 100 previously identified transcriptional regulatory elements. This juxtaposition of structural and regulatory elements of otherwise unrelated genes suggests a mechanism by which the evolutionarily conserved genetic linkage of 21OH-A and slp (or the homologous complement component C4) might provide a selective advantage. Analogous genetic arrangements may explain other examples of conserved linkage of disparate genes.

The murine Slp gene. Additional evidence that sex-limited protein has no biologic function.

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.

Differential expression of the five C4-related genes of H-2 w7mice

Mice bearing the H-2w7 haplotype have five C4-related genes, one C4, one Slp, and three C4/Slp hybrid genes. The expression of these five genes in the liver of H-2w7 mice was estimated at the steady state level of their respective mRNA. We have amplified by the polymerase chain reaction (PCR) three regions of the C4/Slp mRNA where some of these five genes show nucleotide substitution. A relative amount of each gene product was estimated by single-strand conformation polymorphism (SSCP) analysis or by direct counting of the number of respective clones after subcloning into a plasmid vector. A steady state level of the C4 mRNA was most abundant among C4-related gene transcripts. The hybrid 1 and 3 genes were expressed at a similar level which is about 1/2-1/3 of the C4 level. The hybrid 2 gene was expressed at about 1/5 of the hybrid 1 or 3 level. Neither male nor female H-2w7 mice expressed the Slp gene. These results showed that the expression of the five C4-related genes of H-2w7 mice is differentially regulated in spite of the close similarity in the nucleotide sequences in both the 5' flanking and coding regions of these genes.

Three extra copies of a C4-related gene in H-2 w7 mice are C4/Slp hybrid genes generated by multiple recombinational events

Mice bearing the H-2w7 haplotype have five C4-related genes and constitutively express the Slp antigen. To understand the structure and evolution of the five C4-related genes of the C3H.W7 mouse, we have determined nucleotide sequences of the 5' end region of these genes. A C4/Slp hybrid nature was confirmed for three of five C4-related genes as predicted previously by restriction enzyme analysis. The nucleotide sequences of the 5' flanking regions of these three hybrid genes showed close similarity to that of the C4 gene, while the 3' side of the ninth exon of the three hybrid genes showed close similarity to that of the Slp gene. In contrast, the regions between the first exon and the middle of the ninth exon of the three hybrid genes showed a mosaic structure of C4-like and Slp-like sequences. Moreover, the boundaries of the C4-like and Slp-like sequences were quite different among the three hybrid genes. The pattern of nucleotide sequence diversity in this region among the five C4-related sequences could be mainly explained not by point mutations but by gene conversions or unequal crossovers. These results suggest that multiple genetic recombinational events between two homologous sequences played an important role in the generation and diversification of the extra copies of the C4/Slp gene in the H-2w7 mouse.

Specific binding of a hepatoma nuclear factor to the NF.kappa B/H2TF1 recognition motif found in the C4 promoter, but not in the Slp promoter.

The fourth component of C (C4) and sex-limited protein (Slp) genes of FM strain mice show more than 95% nucleotide identity both in the coding and the about 2 kb of 5' flanking region, but are distinct in their regulation. Although C4 is a constitutive gene, Slp is an androgen-dependent gene and shows negligible basal level expression. We have previously shown that this difference in basal expression is determined by a region of about 400 bp immediately 5' of the transcriptional start site of the C4 and Slp genes. In this report, we demonstrate a specific binding site for a HepG2 nuclear extract in this region of the C4 gene by gel retardation and DNase I footprinting experiments. Nucleotide sequence of this binding site is very similar to the recognition sequence of well characterized transcription factors, NF.kappa B and H2TF1. Synthetic oligonucleotides representing the binding sites of the H-2Kb class I and IL-6 genes for these factors compete with the C4 promoter for the complex formation with a HepG2 nuclear factor. Due to the nucleotide deletion and substitution, the corresponding region of the Slp gene lacks this sequence motif as well as binding activity to the HepG2 nuclear factor.

Mapping of the transcriptional regulatory domains responsible for the difference in the promoter activity between mouse C4 and Slp (sex-limited protein) genes.

The genes for the fourth complement component (C4) and sex-limited protein (Slp) of the mouse exhibit a high degree of sequence homology and have probably been derived by recent gene duplication from a common ancestor gene, but their mode of expression is greatly different in most standard mouse strains. Although the C4 gene is constitutively expressed, the Slp gene is expressed only under the influence of androgens or not expressed at all. To map the transcriptional regulatory domains that are responsible for the difference in the promoter activity between the mouse C4 and Slp genes, we constructed various hybrid promoters from the 5'-flanking DNA of the C4 and Slp genes of the FM strain and tested for their transcriptional activity. The hybrid promoters were inserted into the HindIII site located immediately upstream of the chloramphenicol acetyl transferase (CAT) gene of the plasmid pSVOcat. The expression of CAT was assayed in transiently transfected HepG2 cells and in permanently transfected Ltk- cells. The transcriptional regulatory region responsible for the difference between the C4 and Slp gene expression was mapped within the 5'-flanking region of about 400 nucleotides near the promoters of the C4 and Slp genes. By S1 mapping analysis, it was shown that the CAT activities expressed in transfected cells were due to the transcripts correctly initiated from the promoter of the C4 or Slp genes in the chimeric constructs.

An ancient provirus has imposed androgen regulation on the adjacent mouse sex-limited protein gene

The mouse sex-limited protein ( Slp) gene is dependent on androgen for expression, unlike its homologous neighbor, which encodes the fourth component of complement ( C4). We have found that the extensive identity of Slp and C4 is disrupted by an endogenous provirus inserted 2 kb upstream of Slp. The 5′ LTR of this element corresponds to the previously characterized hormone-responsive enhancer associated with Slp regulation, leading to the conclusion that the provirus has conferred androgen response on the adjacent Slp gene. The provirus is extremely old, based on LTR sequence divergence, the accumulation of mutations in former retroviral-like coding regions, and its stability within the mouse genome. The association of this transposable element with Slp regulation thus provides a long-sought example of an insertional mutation that has been maintained in evolution.

A complex androgen-responsive enhancer resides 2 kilobases upstream of the mouse Slp gene.

Neighboring genes encoding the mouse sex-limited protein (Slp) and fourth component of complement (C4) show extensive homology. In contrast to C4, however, Slp is regulated by androgen. One region of the Slp gene capable of hormonal response following transfection was located about 2 kilobases upstream of the transcription start site, where the C4 and Slp sequences diverge. This region, delimited here to a 0.75-kilobase fragment, showed cryptic promoter activity as well as androgen responsiveness in either orientation in front of the bacterial chloramphenicol acetyltransferase coding region. When this fragment was placed upstream of a viral long terminal repeat, increased chloramphenicol acetyltransferase expression derived from the viral promoter. Proteins from nuclear extracts specifically bound to four sequences within the region, near sites that are DNase I hypersensitive in vivo and reflect the hormonal and developmental regulation of Slp. Like several other cellular enhancers, this androgen-responsive element seems to be modular in nature and complex in its function.

A complex androgen-responsive enhancer resides 2 kilobases upstream of the mouse Slp gene.

Neighboring genes encoding the mouse sex-limited protein (Slp) and fourth component of complement (C4) show extensive homology. In contrast to C4, however, Slp is regulated by androgen. One region of the Slp gene capable of hormonal response following transfection was located about 2 kilobases upstream of the transcription start site, where the C4 and Slp sequences diverge. This region, delimited here to a 0.75-kilobase fragment, showed cryptic promoter activity as well as androgen responsiveness in either orientation in front of the bacterial chloramphenicol acetyltransferase coding region. When this fragment was placed upstream of a viral long terminal repeat, increased chloramphenicol acetyltransferase expression derived from the viral promoter. Proteins from nuclear extracts specifically bound to four sequences within the region, near sites that are DNase I hypersensitive in vivo and reflect the hormonal and developmental regulation of Slp. Like several other cellular enhancers, this androgen-responsive element seems to be modular in nature and complex in its function.