A chromosome scale assembly of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), genome.

OBJECTIVE: The tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species. RESULTS: The initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions.

Susceptibilities of Helicoverpa zea (Lepidoptera: Noctuidae) Populations From the Mississippi Delta to a Diamide Insecticide.

The corn earworm, Helicoverpa zea (Boddie), is a major pest of row crops in the Southern United States. Control of this insect is dependent on preventative insecticidal transgenic crops and synthetic insecticide applications when damaging populations are encountered in the field. Recently, the use of chemicals from the diamide class of insecticides, particularly chlorantraniliprole, has been used to control unacceptable populations. Due to the increased importance of this active ingredient for control of corn earworms, populations of this insect from the Mississippi Delta have been monitored for susceptibilities annually since 2016. Overall, 58 populations of H. zea were examined for their susceptibility to chlorantraniliprole through diet-incorporated bioassays from 2016 to 2021. Based on probit analysis, there was only a 4-fold difference between the highest and lowest LC50 estimates for all populations tested. Through weights of 2nd and 3rd instar larvae, there appears to be a substantial fitness cost associated with surviving caterpillars that fed on various concentrations of chlorantraniliprole in bioassays, which is not captured through the yes or no response of typical survival analysis. Overall, there was not a detectable trend of reduced susceptibility to chlorantraniliprole over the course of the six-year study.

The digestive system in Zygentoma as an insect model for high cellulase activity.

The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-ß-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group.

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Effectiveness of Microbial and Chemical Insecticides for Supplemental Control of Bollworm on Bt and Non-Bt Cottons.

Laboratory and field experiments were conducted to determine the effectiveness of microbial and chemical insecticides for supplemental control of bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), on non-Bt (DP1441RF) and Bt (DP1321B2RF) cottons. Neonate and 3rd instar larvae survival was evaluated on leaf tissue treated with microbial and chemical insecticides including a commercial formulation of Bacillus thuringiensis (Dipel), a Heliothis (Helicoverpa) nuclear polyhedrosis virus (NPV; Gemstar), λ-cyhalothrin (Karate Z), and chlorantraniliprole (Prevathon). Residual activity of insecticides was measured in a small plot field experiment. The performance of microbial insecticides, with the exception of a mid-rate of Dipel with neonate larvae, was comparable with that of chemical treatments on non-Bt cotton leaves with regard to 1st and 3rd instar bollworm mortality at 10 d and pupal eclosion at 20-d post treatment. Production-level field evaluations of supplemental bollworm control in non-Bt and Bt cottons with NPV, λ-cyhalothrin, and chlorantraniliprole were also conducted. During both years of the field study, all chemical and microbial treatments were successful in suppressing bollworm larval densities in non-Bt cotton below economic threshold levels. Overall, net returns above bollworm control, regardless of treatment, were negatively correlated with larval abundance and plant damage. In addition, there was no economic benefit for supplemental control of bollworms in Bt cotton at the larval densities observed during this study. These data provide benchmark comparisons for insect resistance management with microbial and chemical insecticides in Bt and non-Bt cottons and strategic optimization of the need to spray non-Bt and Bt cotton in IRM programs.

Expression profiles of astakine-like transcripts in the tarnished plant bug, Lygus lineolaris, exposed to fungal spores of Beauveria bassiana.

Astakines are hematopoietic cytokines originally isolated from crustaceans. We identified three astakine-like transcripts in the tarnished plant bug (Lygus lineolaris), LlAst-1, LlAst-2 and LlAst-3, containing prokineticin domains. Quantitative real-time PCR showed variation in expression patterns of astakines in different tissues and between sexes. Relative expression levels of LlAst-1 were highest in the fat bodies of females, while LlAst-2 expression was highest in the fat bodies of both males and females. LlAst-3 expression was higher in male legs compared with the female legs, but lower in all other tissues. Infection with the entomopathogenic fungus Beauveria bassiana slightly elevated LlAst-1 expression 48 h after infection in both males and females. In contrast, the expression levels of LlAst-2 and LlAst-3 were not significantly changed in males and females. Compared with 12:00 h, LlAst-1 level was higher in both sexes at 18:00 h and 00:00 h (midnight). By 6:00 h, the LlAst-1 level in females was significantly reduced while that in males remained high. LlAst-2 and -3 had highest relative expression levels in females at midnight but were significantly lower than in males at midnight and in both sexes at 18:00 h and 6:00 h. This is the first report of expression of astakine-like cytokines from insects.

Cloning and expression profiling of odorant-binding proteins in the tarnished plant bug, Lygus lineolaris.

In insects, the perception and discrimination of odorants requires the involvement of odorant-binding proteins (OBPs). To gain a better molecular understanding of olfaction in the agronomic pest Lygus lineolaris (the tarnished plant bug), we used a transcriptomics-based approach to identify potential OBPs. In total, 33 putative OBP transcripts, including the previously reported Lygus antennal protein (LAP), were identified based on the characteristic OBP Cys signature and/or sequence similarity with annotated orthologous sequences. The L. lineolaris OBP (LylinOBP) repertoire consists of 20 'classic' OBPs, defined by the spacing of six conserved Cys residues, and 12 'Plus-C' OBPs, defined by the spacing of eight conserved Cys and one conserved Pro residue. Alternative splicing of OBP genes appears to contribute significantly to the multiplicity of LylinOBP sequences. Microarray-based analysis of chemosensory tissues (antennae, legs and proboscis) revealed enrichment of 21 LylinOBP transcripts in antennae, 12 in legs, and 15 in proboscis, suggesting potential roles in olfaction and gustation respectively. PCR-based determination of transcript abundance for a subset of the LylinOBP genes across multiple adult tissues yielded results consistent with the hybridization data.

Effect of food and temperature on emergence from diapause in the tarnished plant bug (Hemiptera: Miridae).

Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), overwinter as diapausing adults in North America. Overwintering adults were collected near Stoneville, MS from blooming henbit, Lamium amplexicaule L., and from plant debris during December and January and dissected to determine their reproductive status. Averaged over four winters, male and female tarnished plant bugs collected from henbit terminated diapause at a significantly higher rate than males and females from plant debris during each week of December and the first week of January. Both sexes in each habitat were nearly all reproductive by the end of January. Adults overwintering in plant debris terminated diapause during January in the absence of a food stimulus in all 5 yr studied. This emergence was thought to be controlled by an internal clock. Laboratory and field studies showed that emergence from diapause could be influenced by food, sex, and temperature. Adults overwintering on a suitable food source, blooming henbit, terminated diapause during December in the 4 yr studied, and males terminated diapause more rapidly than females. Food quality was important in emergence from diapause, and females on blooming henbit terminated diapause at a significantly higher rate than females on nonblooming mustard, Brassica juncea (L.) Cosson. Laboratory tests showed that diapausing adults reared in the laboratory and held at a diapause-maintaining photoperiod of 10:14 (L:D) h could be terminated from diapause by using food and temperature stimuli. The lower thermal threshold for development to reproductive adults was found to be near 10°C. The ability of diapausing adults to respond to food and temperature stimuli in December can enable tarnished plant bugs to take advantage of warm winters and winter hosts to produce a new generation earlier.

Offspring from sequential matings between Bacillus thuringiensis-resistant and Bacillus thuringiensis-susceptible Heliothis virescens moths (Lepidoptera: Noctuidae).

The tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is an economically important pest of the Americas. Females of this species copulate multiple times during their lifetimes, and the presence of sperm from multiple males inside them could allow for a diversity of paternal genotypes in the offspring, unless there was complete precedence of sperm from the first mating. If a female copulates with a male that is insecticide-susceptible and another male that is insecticide-resistant, her progeny could vary in their resistance phenotypes. In some cases, this could impact the evolution of insecticide resistance in a population. We designed a series of experiments to determine whether Bacillus thuringiensis susceptibility is maintained when an H. virescens female that is homozygous for a genetically recessive form of B. thuringiensis resistance copulates with a Cry1Ac-susceptible and a Cry1Ac-resistant males. During the lifetime of double-copulated females, a proportion of F1 progeny were Cry1Ac-resistant. This indicates that when a B. thuringiensis-resistant H. virescens female copulates with two males, with one male being resistant to Cry1Ac, some of the progeny will carry resistance to this insecticide. Due to the polyandrous nature of this species, the above-mentioned scenario is not unrealistic; therefore, results from this study may help understand and manage the evolution of B. thuringiensis-resistance in field populations.

Utilization of early soybeans for food and reproduction by the tarnished plant bug (Hemiptera: Miridae) in the delta of Mississippi.

Commercially produced maturity group (MG) IV soybeans, Glycine max L., were sampled during bloom for tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), during May and June 1999 (3 fields) and 2001 (18 fields). The adults and nymphs were found primarily in single population peaks in both years, indicating a single new generation was produced during each year. The peak mean numbers of nymphs were 0.61 and 0.84 per drop cloth sample in 1999 and 2001, respectively. Adults peaked at 3.96 (1999) and 3.76 (2001) per sweep net sample (25 sweeps). Tests using laboratory-reared and field-collected tarnished plant bugs resulted in very poor survival of nymphs on 16 different soybean varieties (MG III, one; IV, four; V, nine; VI, two). A large cage (0.06 ha) field test found that the number of nymphs produced on eight soybean varieties after mated adults were released into the cages was lower than could be expected on a suitable host. These results indicated that soybean was a marginal host for tarnished plant bugs. However, the numbers of adults and nymphs found in the commercially produced fields sampled in the study may have been high enough to cause feeding damage to the flowering soybeans. The nature of the damage and its possible economic importance were not determined. Reproduction of tarnished plant bugs in the commercially produced early soybean fields showed that the early soybeans provided tarnished plant bugs with a very abundant host at a time when only wild hosts were previously available.

Response of Heliothis virescens (Lepidoptera: Noctuidae) strains to Bacillus thuringiensis Cry1Ac incorporated into different insect artificial diets.

Susceptibility to the Cry1Ac toxin from Bacillus thuringiensis in tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is usually measured by performing bioassays under laboratory conditions. Accurate comparison of Cry1Ac susceptibility among H. virescens samples conducted in different places is challenged by several important methodological aspects, especially if different insect artificial diets are used to perform bioassays. In this study, we compared Cry1Ac susceptibility of four different-origin H. virescens colonies when challenged with this toxin incorporated into four different insect artificial diets. Our data show that Cry1Ac susceptibility was lower in all the H. virescens colonies for one of the commercial diets (Bio-Serv). Bio-Serv diet was one of the least significantly consumed diets by larvae of the four different colonies, which indicates that insects encountered less Cry1Ac toxin due to lower consumption of diet. Larvae fed Bio-Serv diet also seemed to display slower Cry1Ac toxin activation compared with larvae fed any of the other three diets tested. In contrast, a wheat germ-soybean diet (ARS) was one of the most consumed diets by the four H. virescens colonies. The increased consumption of ARS diet probably led to the high level of Cry1Ac susceptibility observed in all the H. virescens colonies. Our data highlight the importance of using common diets and use a standard tobacco budworm colony when comparing Cry1Ac susceptibility between diverse H. virescens strains or across time.

Bacillus thuringiensis Cry1Ac resistance frequency in tobacco budworm (Lepidoptera: Noctuidae).

The tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is one of the most important pests of cotton, Gossypium hirsutum L, that has become resistant to a wide range of synthetic insecticides. CrylAc-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the constant exposure of tobacco budworm to this protein toxin may result in the development of resistance to it. To estimate the frequency of alleles that confer resistance to a 1.0 microg of Bacillus thuringiensis Cry1Ac diagnostic concentration in field-collected insects, the second generation (F2) of 1,001 single-pair families from seven geographical regions representing 2,202 alleles from natural populations was screened in 2006 and 2007 without finding major resistant alleles. Neonates of 56 single-pair families were able to develop to second instar on the diagnostic concentration in the initial screen, but only seven of these lines did so again in a second confirmatory screen. Minor resistance alleles to Cry1Ac may be quite common in natural populations of H. virescens. Our estimated resistance allele frequencies (0.0036-0.0263) were not significantly different from a previously published estimate from 1993. There is no evidence that H. virescens populations have become more resistant to Cry1Ac.

An empirical test of the F2 screen for detection of Bacillus thuringiensis-resistance alleles in tobacco budworm (Lepidoptera: Noctuidae).

Insects exposed to genetically modified crops expressing Bacillus thuringiensis (Bt) toxins are under intense selection pressure that could result on widespread Bt resistance. Screening for early indications of Bt resistance developing in targeted Lepidoptera is conducted in many of the regions where genetically modified cotton and corn have been commercialized. Heliothis virescens (F.) (Lepidoptera: Noctuidae) has been selected in the laboratory to have a gene for resistance to Cry1Ac. We used this laboratory line to test the assumptions and theoretical predictions related to detection of recessive Bt-resistant alleles in field populations based on a second generation (F2) screen. By creating single-pair families from mating a heterozygous Cry1Ac-resistant moth with a Cry1Ac-susceptible moth, we simulated the most common genotype when Bt-resistance alleles are at low frequency in the field. The second generation (F2) neonates of single-pair families were screened daily with diagnostic concentration bioassays. Cry1Ac-resistant homozygous larvae were detected, but the proportion of resistant larvae was generally below the theoretical expectation of 6.25% and was influenced by the moth F1 sib-mating density and by the day of oviposition of F2 eggs. Logistical considerations such as F1 sib-mating density and F2 neonate screening are important for the successful implementation of a reliable method.

Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient.

Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.

cDNA of YP4, a follicular epithelium yolk protein subunit, in the moth, Plodia interpunctella.

YP4, a subunit of the follicular epithelium yolk protein in the moth, Plodia interpunctella, is produced in the follicle cells during vitellogenesis and after secretion is taken up into the oocyte and stored in the yolk spheres for utilization during embryogenesis. In order to identify the cDNA clones for YP4, a degenerate PCR primer was designed to six amino acid residues identified in the NH2-terminal sequence of mature YP4. The YP4 degenerate primer plus T7 reverse PCR primer produced a PCR product from a cDNA library for the majority of the YP4 coding sequence. Combined cDNA and 5' RACE sequencing showed the YP4 transcript to be 991 bp in length with a single open reading frame for a predicted polypeptide of 299 amino acids. Northern analysis showed a single YP4 transcript was present in ovarian RNA that was approximately 1 kb in length. The predicted amino acid sequence for YP4 from P. interpunctella was most closely related to the predicted YP4 protein from the moth, Galleria mellonella, and the spherulin 2a protein from the slime mold, Physarum polycephalum.

Species-specific repeat units in the intergenic spacer of the ribosomal RNA cistron of Anopheles aquasalis Curry.

A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.

alpha-Crystallin protein cognates in eggs of the moth, Plodia interpunctella: possible chaperones for the follicular epithelium yolk protein.

alpha-Crystallin protein cognates were found in germ cells of the Indianmeal moth, Plodia interpunctella (Shirk and Zimowska, 1997). A cDNA clone of 674 bp with a single open reading frame was isolated for a 25,000 molecular weight polypeptide member of this family, alpha CP25, and a single transcript of approximately 700 bp was found in the ovary of vitellogenic females. Both the DNA sequence and predicted amino acid sequence showed considerable homology with the embryonic lethal gene, l(2)efl, in Drosophila melanogaster. In addition to the sequence for l(2)efl, the predicted amino acid sequence for acp25 also showed significant sequence similarly with the alpha-crystallin A chain polypeptides from the lenses of vertebrae eyes. An N-terminal hydrophobic aggregation site and a C-terminal protective binding site common to alpha-crystallin proteins were present in the predicted acp25 and l(2)efl amino acid sequences, while only the C-terminal protective binding site was present in the small heat shock protein sequences from D. melanogaster. This evidence suggests that although the alpha-crystallin protein cognates in P. interpunctella evolved from a gene common with small heat shock protein genes, the amino acid sequence has converged on a structure similar to that of alpha-crystallin proteins. Native immunoblot analysis showed that the alpha-crystallin proteins formed high molecular weight complexes with the follicular epithelium yolk protein (FEYP) but not vitellin in yolk. An electroblot binding assay was used to show that the germ-cell alpha-crystallins of P. interpunctella bind specifically with the FEYP and that the binding was reversible in the presence of ATP or low pH. This evidence in conjunction with the evidence that the alpha-crystallins and FEYP form a stable complex that co-purifies from native egg proteins suggests that the alpha-cystallin cognates function as chaperones for the follicular epithelium yolk proteins in the embryos of P. interpunctella.

5' coding region of the follicular epithelium yolk polypeptide 2 cDNA in the moth, Plodia interpunctella, contains an extended coding region.

The 5' region of YP2 cDNA, a follicular epithelium yolk protein subunit in the moth, Plodia interpunctella, shows that the polypeptide contains an extended internal coding region. Partial cDNA clones for YP2 were isolated from a pharate adult female ovarian cDNA expression library in Lambda Zap II by screening with antigen selected YP2 antiserum. The 5' sequence of the YP2 transcript was determined by 5' RACE PCR of ovarian mRNA using YP2 sequence-specific nested primers. The combined cDNA and 5' RACE sequencing showed the YP2 transcript to be 1971 bp in length up to the poly(A) tail with a single open reading frame for a predicted polypeptide of 616 amino acids. Northern analysis showed a single YP2 transcript to be present in ovarian RNA that was approximately 2 kb in length. The predicted amino acid sequence for YP2 from P. interpunctella is most closely related to egg specific protein (ESP) from Bombyx mori and the partial YP2 sequence from Galleria mellonella. YP2 from P. interpunctella also is similar to vertebrate lipases and contains a conserved lipid binding region. However, the 5' coding region of YP2 from P. interpunctella contains an in-frame insert of approximately 438 bp that had replaced an approximately 270-bp region as compared with ESP from B. mori and YP2 of G. mellonella. This suggests that the insert occurred by a recombinational event internal to the YP2 structural gene of P. interpunctella.

Multiple upstream promoter elements of the gene for the pregnancy-associated tartrate-resistant acid phosphatase, uteroferrin bind human endometrial nuclear proteins.

The endometrial expression of the gene encoding porcine uteroferrin (UF), during pregnancy is presumed to be mediated by cis-regulatory regions distinct from those that confer its limited expression to other mammalian tissues and cell types. In the present study, chimeric DNA constructs of native and progressive 5' deleted promoter regions fused to the promoter chloramphenicol acetyl-transferase reporter gene were transiently transfected in the human endometrial carcinoma cell line ECC-1 to examine their ability to direct UF promoter activity. The region between -1935 and -831 bp contained negatively acting elements which drastically reduced basal promoter activity. In contrast, the region between -831 and -484 bp contributed significantly to high level basal activity. Gel retardation and footprinting assays identified factor-binding sites between -1601 and -484 bp for human endometrial nuclear proteins. One binding site corresponds to a heptamer motif (TGCTAGA) present twice within the -1601 to -831 bp region and previously shown to bind an 80 kDa porcine endometrial protein. This heptamer bound an 80 kDa nuclear protein from human ECC-1 and human Ishikawa endometrial cells and a 92 kDa protein from human placental JEG-3 cells. The other binding region within -831 to -484 bp contained GC-rich sequences, which bind human Sp1. The protected GC-rich sequence (GC-Box 1) between -768 and -749 bp also binds a 24 kDa M(r) protein. Nuclear proteins of molecular weight 40-60 kDa and distinct from Sp1, Sp2 and Sp3 bound a second GC-rich sequence (GC-Box 3) between -628 and -616 bp. These studies demonstrate that multiple elements within the UF gene promoter bind nuclear proteins which are similarly expressed in other endometrial cells and suggest that common transactivating factors may functionally mediate expression of endometrial-associated genes.