Pentanucleotide Sequence Research Articles

A pyrazolopyridine as a novel AhR signaling activator with anti-breast cancer properties in vitro and in vivo

Breast cancer is one of the main causes of malignancy-related deaths globally and has a significant impact on women's quality of life. Despite significant therapeutic advances, there is a medical need for targeted therapies in breast cancer. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor mediates responses to environment stimuli, is emerging as a unique pleiotropic target. Herein, a combined molecular simulation and in vitro investigations identified 3-(3-fluorophenyl)-1H-pyrazolo[3,4-b]pyridine (3FPP) as a novel AhR ligand in T47D and MDA-MB-231 breast cancer cells. Its agonistic effects induced formation of the AhR-AhR nuclear translocator (Arnt) heterodimer and prompted its binding to the penta-nucleotide sequence, called xenobiotic-responsive element (XRE) motif. Moreover, 3FPP augmented the promoter-driven luciferase activities and expression of AhR-regulated genes encoding cytochrome P450 1A1 (CYP1A1) and microRNA (miR)-212/132 cluster. It reduced cell viability, migration, and invasion of both cell lines through AhR signaling. These anticancer properties were concomitant with reduced levels of B-cell lymphoma 2 (BCL-2), SRY-related HMG-box4 (SOX4), snail family zinc finger 2 (SNAI2), and cadherin 2 (CDH2). In vivo, 3FPP suppressed tumor growth and activated AhR signaling in an orthotopic mouse model. In conclusion, our results introduce the fused pyrazolopyridine 3FPP as a novel AhR agonist with AhR-specific anti-breast cancer potential in vitro and in vivo.

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

Profile and development of microsatellite primers for Acanthogobius ommaturus based on high-throughput sequencing technology

Acanthogobius ommaturus, a fish species of the Family Gobiidae, is a marine commercial fish perched on the bottom of seawater. In this study, Illumina high-throughput sequencing technology was applied to obtain the candidate microsatellite markers of A. ommaturus. A total of 4 746 microsatellite-rich fragments were found, of which 4 542 microsatellites are with primer fragments, containing 971 dinucleotide sequences, 2 643 trinucleotide sequences, 569 tetranucleotide sequences, 406 pentanucleotide sequences, and 212 hexanucleotide sequences. Based on the results of high-throughput sequencing, a total of 141 pairs of the microsatellite primers were designed and screened. And then 24 polymorphic primers were finally obtained by polyacrylamide gel electrophoresis. In total, 271 alleles were detected in the 24 pairs of primers. The number of alleles for different primers ranged from 5 to 19. The average number of effective alleles (Na) was 11.292; the average observed heterozygosity (Ho) of the 24 pairs of primers was 0.665, the average expected heterozygosity (He) was 0.880, and the average polymorphic information content was 0.846. All sites were highly polymorphic (PIC>0.50).

Identification of ISSR and RAPD markers linked to yield traits in bread wheat under normal and drought conditions.

Genetic variability and identification of some molecular markers were studied in twenty promising lines of wheat using agronomic traits, ISSR (inter simple sequences repeats) and RAPD (random amplified polymorphic DNA) markers. Significant variation was evidenced in all agronomic traits. The lines proved to be superior to the check cultivar Sahel1 in yield and its component traits. Lines L2, L7 and L8 were the best in most yield component traits in both seasons. Moreover, Lines L2, L4, L5, L7 and L8 showed drought tolerance by which they displayed high performance in agronomic traits as well as a low drought susceptibility index. The percentage of polymorphism was 39.3% and 53.2% for ISSRs and RAPDs, respectively. UBC-881 belonged to penta-nucleotide repeat sequences (GGGTG) that produced the highest level of polymorphism, while UBC-846 belonged to di-nucleotide repeat sequences (CA) that produced the lowest level of polymorphism. Genetic similarities among wheat lines based on ISSR and RAPD markers ranged from 0.81 to 1.00 and from 0.86 to 0.98, respectively. There was a low average of PIC (polymorphism information content) values which were 0.10 (ISSR) and 0.15 (RAPD). The RAPD technique exhibited a higher marker index (MI = 0.69) compared to ISSR (MI = 0.43). There was insignificant correlation between ISSR and RAPD data (0.168, p > 0.05). There were two markers (UBC-881450bp and OPF-10540bp), on each of which two traits regressed significantly. The associated markers each explained a maximum regression of 18.92–34.95% of the total available variation for individual associated traits.

Isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (Hypsipetes amaurotis) in Japan.

Five species, Mammalian orthoreovirus, Avian orthoreovirus (ARV), Nelson Bay orthoreovirus (NBV), Baboon orthoreovirus and Reptilian orthoreovirus, have been identified in the genus Orthoreovirus. Their genomes each consist of 10 dsRNA segments. A novel orthoreovirus was isolated from the haemorrhagic intestine of a dead brown-eared bulbul (Hypsipetes amaurotis) in Japan. The virus formed syncytia in Caco-2 and Vero cells. Electron microscopy revealed non-enveloped capsids of ~70 nm diameter, which were characteristic of reoviruses. Complete genomic sequences were determined. The S1 segment was tricistronic and encoded three proteins, p10, p17 and σC, as in the two species ARV and NBV. Sequence and phylogenetic analyses showed that the virus was similar to ARV and NBV, but was located on a phylogenetic branch different from that of ARV and NBV. The virus had the closest phylogenetic relationship to two reovirus strains: SSRV from a Steller sea lion in Canada and PsRV Ge01 from a psittaciform bird in Europe. The 10 RNA segments had a 3' pentanucleotide sequence (UCAUC-3') conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal heptasequence (5'-GCUUUUC) that was the same as those of SSRV and PsRV Ge01. These results suggested that the novel virus might form a new species with the two strains in the genus Orthoreovirus.

Generation, functional annotation and comparative analysis of black spruce (Picea mariana) ESTs: an important conifer genomic resource

BackgroundEST (expressed sequence tag) sequences and their annotation provide a highly valuable resource for gene discovery, genome sequence annotation, and other genomics studies that can be applied in genetics, breeding and conservation programs for non-model organisms. Conifers are long-lived plants that are ecologically and economically important globally, and have a large genome size. Black spruce (Picea mariana), is a transcontinental species of the North American boreal and temperate forests. However, there are limited transcriptomic and genomic resources for this species. The primary objective of our study was to develop a black spruce transcriptomic resource to facilitate on-going functional genomics projects related to growth and adaptation to climate change.ResultsWe conducted bidirectional sequencing of cDNA clones from a standard cDNA library constructed from black spruce needle tissues. We obtained 4,594 high quality (2,455 5' end and 2,139 3' end) sequence reads, with an average read-length of 532 bp. Clustering and assembly of ESTs resulted in 2,731 unique sequences, consisting of 2,234 singletons and 497 contigs. Approximately two-thirds (63%) of unique sequences were functionally annotated. Genes involved in 36 molecular functions and 90 biological processes were discovered, including 24 putative transcription factors and 232 genes involved in photosynthesis. Most abundantly expressed transcripts were associated with photosynthesis, growth factors, stress and disease response, and transcription factors. A total of 216 full-length genes were identified. About 18% (493) of the transcripts were novel, representing an important addition to the Genbank EST database (dbEST). Fifty-seven di-, tri-, tetra- and penta-nucleotide simple sequence repeats were identified.ConclusionsWe have developed the first high quality EST resource for black spruce and identified 493 novel transcripts, which may be species-specific related to life history and ecological traits. We have also identified full-length genes and microsatellite-containing ESTs. Based on EST sequence similarities, black spruce showed close evolutionary relationships with congeneric Picea glauca and Picea sitchensis compared to other Pinaceae members and angiosperms. The EST sequences reported here provide an important resource for genome annotation, functional and comparative genomics, molecular breeding, conservation and management studies and applications in black spruce and related conifer species.

Complete sequence of a reovirus associated with necrotic focus formation in the liver and spleen of Muscovy ducklings

The complete sequence of a reovirus, strain 815-12 associated with necrotic focus formation in the liver and spleen of Muscovy ducklings in China, was determined and compared with sequences of other duck-, goose-, and chicken-origin reoviruses. The 815-12 genome comprised 22,969 bp with 10 dsRNA segments ranging from 1125 bp (S4) to 3958 bp (L1), all of which (except S4) were almost identical in length to the cognate segments of other waterfowl and chicken isolates. Detailed analyses revealed that 815-12 and other waterfowl isolates contained the conserved 3'-terminal pentanucleotide sequence (UCAUC-3') of the orthoreoviruses and 5'-terminal hexanucleotide sequence (5'-GCUUUU) of avian orthoreoviruses (ARVs), and conserved functional motifs previously identified in ARV proteins. Several notable differences, including organization of the polycistronic genome segments and genomic coding assignments of the S segments, existed between viruses represented by 815-12 and the waterfowl reoviruses emerging in China in recent years; the latter was somewhat similar to chicken isolates. Pairwise sequence comparisons demonstrated extensive sequence diversity among the various waterfowl isolates and between waterfowl and chicken isolates. Phylogenetic analyses identified two genetic groups for waterfowl reoviruses, and potential genetic reassortment of segment M2 between waterfowl and chicken reoviruses and segments encoding for λA, λB, μA, μNS and σA between waterfowl reoviruses. Taken together, it was suggested that common designation ARV-Wa should be used to represent ARV isolates from different waterfowl species and that the two ARV-Wa genotypes should be considered as two separate groups distinct from chicken isolates within the species Avian orthoreovirus.

Replacement of conserved or variable sequences of the mosquito-borne dengue virus 3′ UTR with homologous sequences from Modoc virus does not change infectivity for mosquitoes

The genus Flavivirus includes both vector-borne and no known vector (NKV) species, but the molecular determinants of transmission mode are not known. Conserved sequence differences between the two groups occur in 5' and 3' UTRs. To investigate the impact of these differences on transmission, chimeric genomes were generated, in which UTRs, UTRs+capsid, or the upper 3' UTR stem-loop of mosquito-borne dengue virus (DENV) were replaced with homologous regions from NKV Modoc virus (MODV); the conserved pentanucleotide sequence (CPS) was also deleted from the DENV genome. Virus was not recovered following transfection of these genomes in three different cell types. However, DENV genomes in which the CPS or variable region (VR) of the 3' UTR were replaced with MODV sequences were recovered and infected Aedes aegypti mosquitoes with similar efficiencies to DENV. These results demonstrate that neither vector-borne CPS nor VR is required for vector-borne transmission.

Toward reading the sequence of short oligonucleotides from their retention factors obtained by means of hydrophilic interaction chromatography and ion‐interaction reversed‐phase liquid chromatography

Retention characteristics of selected synthetic 5'-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.

Non-CpG Methylation Occurs in the Regulatory Region of the Sry Gene

The Sry (sex determining region on Y chromosome) gene is a master gene for sex determination. We previously reported that the Sry gene has tissue-dependent and differentially methylated regions (T-DMRs) by analyzing the DNA methylation states at CpG sites in the promoter regions. In this study, we found unique non-CpG methylation at the internal cytosine in the 5'-CCTGG-3' pentanucleotide sequence in the Sry T-DMR. This non-CpG methylation was detected in four mouse strains (ICR, BALB/c, DBA2 and C3H), but not in two strains (C57BL/6 and 129S1), suggesting that the CCTGG methylation is tentative and unstable. Interestingly, this CCTGG methylation was associated with demethylation of the CpG sites in the Sry T-DMR in the developmental process. A methylation-mediated promoter assay showed that the CCTGG methylation promotes gene expression. Our finding shows that non-CpG methylation has unique characteristic and is still conserved in mammals.

Novel aspects of heat shock factors: DNA recognition, chromatin modulation and gene expression

Heat shock factor (HSF) is an evolutionarily conserved stress-response regulator that activates the transcription of heat shock protein genes, whose products maintain protein homeostasis under normal physiological conditions, as well as under conditions of stress. The promoter regions of the target genes contain a heat shock element consisting of multiple inverted repeats of the pentanucleotide sequence nGAAn. A single HSF of yeast can bind to heat shock elements that differ in the configuration of the nGAAn units and can regulate the transcription of various genes that function not only in stress resistance, but also in a broad range of biological processes. Mammalian cells have four HSF family members involved in different, but in some cases similar, biological functions, including stress resistance, cell differentiation and development. Mammalian HSF family members exhibit differential specificity for different types of heat shock elements, which, together with cell type-specific expression of HSFs is important in determining the target genes of each HSF. This minireview focuses on the molecular mechanisms of DNA recognition, chromatin modulation and gene expression by yeast and mammalian HSFs.

Why is p53-inducible gene 3 rarely affected in cancer?

Dear Sir, We read with great interest the work on the contribution of the p53-inducible gene 3 (PIG3) to the early cellular response to DNA damage (Lee et al., 2010). PIG3 was considered until now as a p53dependent proapoptotic gene mediating reactive oxygen species (ROS) production. p53 regulates PIG3 through a polymorphic pentanucleotide sequence. An individual’s constitution or deregulation of this repetitive sequence, by microsatellite instability (MSI), was postulated as potential mechanisms for cancer susceptibility (Contente et al., 2002). Collectively, we and others (Gorgoulis et al., 2004 and references therein), along with our additional unpublished data from 114 lung tumors (7% loss of heterozygosity (LOH) and 0.9% MSI), have shown that MSI and LOH rarely affect this locus in various common tumors, whereas rare pentanucleotide alleles are not associated with cancer risk. An alternative splicing of PIG3 mRNA resulting in an unstable truncated variant (Nicholls et al., 2004) could represent another PIG3 inactivation mechanism. Nevertheless, our unpublished work in a subset of the previously mentioned lung tumors for potential PIG3 alternative splicing deregulation favoring the truncated form was not found. On contrary, the full-length transcript was predominantly found to be increased in tumor counterparts (96.7%). Collectively, these results raise the question: why is PIG3 rarely affected in cancer? Lee et al. (2010) provide new insights into the role of PIG3 and potentially the answer to the above question. Its presence in the nucleus, in which it functions as an upstream component of the DNA damage response (DDR) pathway, suggests that like other components, similarly located in this pathway, it must be intact. Upstream DDR components are rarely altered, in contrast to p53, as cancer cells are likely to rely on them to optimize the survival (Negrini et al., 2010 and references therein). Even more, p53 also regulates PIG3 expression thus creating a potential positive feedback with the upstream DDR mechanism, suggesting that in the case of DNA damage p53 can also amplify directly the upstream response in normal cells. This may be valid for tumor cells also, given that certain p53 mutants activate PIG3, whereas the result of our study show increased full-length transcript expression in tumor counterparts. Therefore, p53 inactivation, as frequently seen in most types of tumors, is sufficient to ‘compromise’ this pathway, with the upstream components remaining intact as they may be vital for tumor cells. Finally, the cytoplasmic role of PIG3 as an ROS producer must not be altered also, as it may be involved in hypoxiamediated tumor clone selection, as previously proposed (Gorgoulis et al., 2004).

Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat

This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3' terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13-50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.

Spinocerebellar ataxia type 31

Spinocerebellar ataxia type 31 (SCA31) is a relatively common degenerative ataxia in Japan. We recently discovered SCA31 mutation as a complex pentanucleotide repeat containing (TAAAA)(n), (TAGAA)(n), and (TGGAA)(n). The size of this repeat ranged from 2.8 to 3.5 kilo-base pairs (kb). Among these repeats, (TGGAA)(n) repeat appears crucial for SCA31 pathogenesis. The length of this complex repeat inversely correlated with ages of onset in patients. The mutation lies in an intron shared by two different genes, BEAN (brain expressed, associated with NEDD4) and TK2 (thymidine kinase 2), which are transcribed in opposite directions. Thus, the complex pentanucleotide sequence is predicted to be transcribed in both directions, but not necessarily translated into proteins. In situ hybridization analysis in patients' Purkinje cells demonstrated that pentanucleotide repeats transcribed in BEAN direction form RNA aggregates ("RNA foci"). We further found that splicing factors, SFRS1 and SFRS9, binds to (UGGAA)(n), the transcript of (TGGAA)(n) in vitro. These findings may imply that SCA31 conforms to pathogenic mechanisms underlying non-coding repeat disorders, such as myotonic dystrophies (DM1 & DM2), and that SFRS1 and SFRS9 are involved in SCA31 pathogenesis.

Genetic Diversity in Spring Wheat Landraces from Northwest of Iran Assessed by ISSR Markers

Inter Simple Sequence Repeat (ISSR) markers were used to study the genetic diversity of 18 spring growth type landraces from Iran and selected 9 cultivars grown in Iran out of 15 ISSR primers .11 primers were found to have enough polymorphism and they were used for assessment. These primers are composed of di, tetra and penta-nucleotide sequences. From a total of 108 DNA fragments produced, 78 (72.22%) fragments were polymorphic. The UPGMA clustering algorithm classified the varieties into three major groups .Majority of landraces located in each group were originated from common locations. The results revealed that ISSR markers could be efficiently used to evaluate genetic variation in the wheat germplasm. Genetic similarity and dissimilarities among genotypes will be useful for genetic differentiation of wheat accessions, selection strategies and genetic development of crop plants.

Molecular characteristics of pharyngeal and invasive emm3 Streptococcus pyogenes strains from Norway, 1988–2003

A major virulence factor of group A streptococci (GAS) is the M protein. Strains with the M3 type are more often associated with necrotizing fasciitis (NF) and streptococcal toxic shock syndrome, and have a higher case fatality rate than strains of other M types. To better understand the epidemiology of M3 GAS strains in Norway, we analyzed 59 invasive and 69 pharyngeal isolates with respect to prophage content, allelic variation in emm3, mtsR encoding the metal transporter of Streptococcus repressor (mtsR), and sclB coding for streptococcal collagen-like protein B. The Norwegian emm3 strains were very homogeneous, mainly harboring the emm allele 3.1 and prophage profile PhiG3.01. Other prophage profiles were transient. The mutation in mtsR known to truncate the protein and result in decreased capacity to cause NF was not found in our isolates. The sclB gene usually harbored five or eight contiguous repeats of a CAAAA pentanucleotide sequence and a highly modular and variable collagen structural motif (CSM) region with 9 and 12 amino acid M3-specific conserved motif repeats distributed across the entire CSM region. Strains with 5 CAAAA repeats emerged in 1993 and these strains were associated with the increase in invasive M3 cases in the period 1993-2003.

The Minimum Replication Origin of Merkel Cell Polyomavirus Has a Unique Large T-Antigen Loading Architecture and Requires Small T-Antigen Expression for Optimal Replication

Merkel cell polyomavirus (MCV) is a recently discovered human polyomavirus causing the majority of human Merkel cell carcinomas. We mapped a 71-bp minimal MCV replication core origin sufficient for initiating eukaryotic DNA replication in the presence of wild-type MCV large T protein (LT). The origin includes a poly(T)-rich tract and eight variably oriented, GAGGC-like pentanucleotide sequences (PS) that serve as LT recognition sites. Mutation analysis shows that only four of the eight PS are required for origin replication. A single point mutation in one origin PS from a naturally occurring, tumor-derived virus reduces LT assembly on the origin and eliminates viral DNA replication. Tumor-derived LT having mutations truncating either the origin-binding domain or the helicase domain also prevent LT-origin assembly. Optimal MCV replication requires coexpression of MCV small T protein (sT), together with LT. An intact DnaJ domain on the LT is required for replication but is dispensable on the sT. In contrast, PP2A targeting by sT is required for enhanced replication. The MCV origin provides a novel model for eukaryotic replication from a defined DNA element and illustrates the selective pressure within tumors to abrogate independent MCV replication.

Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae)

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.

Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication

The pentanucleotide (PN) sequence 5'-CACAG-3' at the top of the 3' stem-loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.