The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene

Genetic variations in ovulation rate which occur in different breeds of sheep provide useful models to explore the mechanisms regulating the development of antral follicles. The Booroola gene, an autosomal mutation that affects ovulation rate, has been known for over two decades and despite intensive research it has not yet been identified. Using resources from human genome mapping and known data about gene linkage and chromosome location in the sheep, we selected the gene encoding the Bone Morphogenetic Protein receptor (BMPR) type 1 B (ALK-6) as a candidate site for the mutation. The BMPR1B gene in the human is located at the region linked with the Booroola mutation, syntenic to chromosome 6 in the sheep. A fragment of the sheep BMPR1B gene was cloned from an ovarian cDNA and the deduced aminoacid (AA) sequence is over 98% homologous to the known mammalian sequences. cDNA and genomic DNA from 20 Booroola genotypes were screened and two point mutation were found in the kinase domain of the receptor, one at base 746 of the coding region (A in the ++ to a G in FF animals) which results in a change from a glutamine in the wild type to a arginine in the Booroola animals. Another point mutation was identified at position 1113, (C to A) but this mutation does not change the coding aminoacid. The first mutation was confirmed in genomic DNA from 10 ewes from an independent Brazilian flock which segregates the Booroola phenotype. In all instances homozygous FecB gene carrier (n=11) had only the 746 A to G mutation, non gene carriers (n=14) had only the wild type sequence and heterozygote gene carriers (n=5) had both sequences. This mutation in the subdomain 3 of the kinase domain could result in an alteration in the expression and/or phosphorylation of SMADs, resulting in the phenotype characteristic of the Booroola animals which is the 'precocious' development of a large number of small antral follicles resulting in increased ovulation rate.

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Fingerprinting of fecB gene in five Egyptian sheep breeds

Biotechnology in Animal Husbandry ◽

10.2298/bah0904205e ◽

2009 ◽

Vol 25 (3-4) ◽

pp. 205-212 ◽

Cited By ~ 5

Author(s):

Hanafy el ◽

Saadani el

Keyword(s):

Point Mutation ◽

Restriction Enzyme ◽

Base Pair ◽

Genomic Dna ◽

Restriction Site ◽

Wild Type ◽

Specific Primer ◽

Sheep Breeds ◽

Pcr Rflp ◽

Pcr Products

Recently, many aspects of FecB gene, including reproductive endocrinology, organs development and body mass have been studied. FecB has an additive effect on litter size and ovulation. The present investigation was carried out to study polymorphism by forced PCR-RFLP of FecB gene in five Egyptian local sheep breeds and its comparison with other foreign sheep breeds. Genomic DNA was isolated from a total of 100 animals of Egyptian sheep breeds namely Rahmani, Ossimi, Awassi, Barki and Awassi x Barki crossbred. Forced PCR of the FecB gene 190 base pair (bp) was amplified using specific primer designed to introduc a point mutation in the resulting PCR products with FecB carrier sheep containing an AvaII restriction site (G|GACC), whereas products from noncarriers lacked (of) this site. Digestion of FecB gene 190 base pair with AvaII restriction enzyme resulted in non carrier 190 bp band (wild type) in all the animals belonging to the five Egyptian breeds studied revealing absence of this restriction site in those five breeds. .

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Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months

Journal of General Virology ◽

10.1099/vir.0.19518-0 ◽

2004 ◽

Vol 85 (2) ◽

pp. 369-377 ◽

Cited By ~ 23

Author(s):

Tapani Hovi ◽

Noora Lindholm ◽

Carita Savolainen ◽

Mirja Stenvik ◽

Cara Burns

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Structural Model ◽

Antigenic Drift ◽

Wild Type ◽

Coding Region ◽

Junction Region ◽

Healthy Siblings ◽

The One

Wild-type 1 poliovirus (wtPV1) strains were isolated from two young healthy brothers shortly after arrival in Finland from Somalia in 1993. Twelve (sibling A) and 18 (sibling B) specimens collected over a period of more than 6 months yielded wtPV1. Partial sequences obtained from the one and two earliest isolates from sibling A and B, respectively, were nearly identical, differing from each other by only one or two nucleotides. Subsequently, the virus evolved separately in both siblings so that maximal differences between strains derived from a given subject peaked at 2·2 % for sibling A, at 1·5 % for sibling B and at 2·5 % between the two siblings in the VP1-coding part of the genome. All substitutions in the 150 nt VP1–2A junction region were synonymous, whereas as many as eight of the 31 variable positions in the remaining VP1-coding region encoded amino acid replacements in at least one strain. Probable structural locations of the variable amino acid positions were mapped to the published PV1/Mahoney structural model. Most of the substitutions occurred around the fivefold axis in motifs that are known to be or suspected to be targets of neutralizing antibodies. We suggest that the striking genetic divergence observed between the strains was based on a combination of bottleneck transmission events and antigenic drift during the prolonged period of poliovirus replication.

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A point mutation in the catalytic domain of c-kit induces growth factor independence, tumorigenicity, and differentiation of mast cells

Blood ◽

10.1182/blood.v87.8.3117.bloodjournal8783117 ◽

1996 ◽

Vol 87 (8) ◽

pp. 3117-3123 ◽

Cited By ~ 103

Author(s):

X Piao ◽

A Bernstein

Keyword(s):

Mast Cell ◽

Point Mutation ◽

Kinase Domain ◽

Loss Of Function ◽

Wild Type ◽

Steel Factor ◽

Kit Receptor ◽

Cytoplasmic Kinase Domain

The murine W and Steel loci encode the Kit receptor tyrosine kinase and its ligand, Steel factor, respectively. Loss of function mutations at either the W or Sl loci lead to a variety of pleiotropic developmental defects, including mast cell deficiency and severe macrocytic anemia. In addition to these loss-of-function mutations, gain-of-function mutations in c-kit, leading to constitutive activation of the Kit receptor, have also been identified in both rodent and human mastocytomas. In this study, we have examined the transforming potential and biologic effects of a point mutation that results in substitution of the aspartic acid at codon 814 in the cytoplasmic kinase domain to tyrosine (D814Y) by introducing either wild-type (Kit) or mutant KitD814Y (KDY) cDNA into an interleukin-3-dependent mast cell line IC2. Stimulation of cells expressing the wild-type Kit receptor (IC2/Kit) with Steel factor in vitro resulted in a short-term growth response, whereas IC2/KDY cells were capable of sustained proliferation in a ligand-independent manner. In addition, expression of KDY resulted in the oncogenic transformation of IC2 cells, as determined by colony formation in vitro in the absence of exogenous growth factors and the formation of mastocytomas in vivo in syngeneic DBA/2 mice. Surprisingly, KDY expression in IC2 cells triggered dramatic changes in cell size and the extent of granulation. In addition, KDY induced the expression of mouse mast cell protease-4 (MMCP-4) and MMCP-6. In contrast, neither of these molecular or cellular changes was observed in IC2/Kit cells treated with Steel factor. These results show that the D814Y mutation in the cytoplasmic kinase domain of the Kit receptor induces ligand-independent mast cell growth in vitro, tumorigenicity in vivo, and mast cell differentiation.

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Identifying Drug-Resistant Mutations in Ebf1-Pdgfrb Ph-like Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.1423.1423 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1423-1423 ◽

Cited By ~ 1

Author(s):

Thai Hoa Tran ◽

Jonathan Van Nguyen ◽

Catherine C. Smith ◽

Kathryn G. Roberts ◽

Charles G. Mullighan ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Point Mutation ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Drug Resistant ◽

Wild Type ◽

Full Spectrum

Abstract Background: Advances in cancer genomics have recently identified a particular group of patients who display a gene expression profile (GEP) similar to that of Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) patients in approximately 15% of children and over 25% of young adults with B-ALL; thus known as Ph-like ALL. The latter has a worse prognosis compared to those without the Ph-like GEP with conventional chemotherapy. Recent studies have unraveled the genomic landscape of Ph-like ALL which is characterized by genetic alterations activating kinase signaling pathways predicted to respond to tyrosine kinase inhibitors (TKIs). In light of the remarkable outcomes of Ph+ALL patients through incorporation of TKI, the Children's Oncology Group (COG) ALL Committee is actively working to incorporate dasatinib for Ph-like ALL patients harboring ABL-class kinase fusions (ABL1, ABL2, PDGFRB, CSF1R) and eventually, ruxolitinib for those with lesions that are predicted to respond to JAK inhibition. While it is hoped that many of these patients will be cured with the addition of relevant TKIs to chemotherapy, we hypothesize that a proportion of patients will develop resistance to TKI, similar to adults with chronic myeloid leukemia who have been treated with long-term TKI. Hence, investigating the underlying mechanisms governing therapy resistance in Ph-like ALL becomes critical for proactively developing novel therapeutic strategies in the relapsed setting. Objectives: To identify the full spectrum of mutations conferring resistance to clinically-active TKIs in Ph-like ALL and to characterize their relative biochemical resistance to different TKIs. Methods: We first focused on the EBF1-PDGFRB rearrangement since this is the most common recurrent kinase-activating fusion genes in pediatric Ph-like ALL. We used a previously validated in-vitro saturation mutagenesis screen to predict the full spectrum of EBF1-PDGFRB drug-resistant mutations. In brief, EBF1-PDGFRB plasmid was propagated into DNA-repair-deficient E. Coli strain XL-1 Red to generate a library of random mutants. Mutagenized EBF1-PDGFRB plasmid was transfected into 293T cells. Viral supernatants were collected and used to infect Ba/F3 cells. Transduced Ba/F3 cells were plated in 1.2% Bacto-agar and exposed to different TKIs (imatinib, dasatinib) at various concentrations. Genomic DNA from IL-3 independent and TKI-resistant colonies was isolated. The PDGFRB kinase domain was amplified and bidirectional sequencing was performed. Results: Our in-vitro screens showed that the vast majority of drug-resistant clones harbor a kinase domain (KD) point mutation. The predominant KD point mutation conferring resistance to imatinib (94%; 168/178 colonies) or dasatinib (81%; 338/416 colonies) was T681I, which is analogous to BCR-ABL1 T315I gatekeeper mutation. N666S was the second most common KD mutation (6%; 18/321 colonies). The full panel of KD mutations recovered is shown in Table 1. Ba/F3 cells harboring mutant EBF1-PDGFRB T681I was 100 times more resistant to dasatinib compared to wild-type and could be rescued by ponatinib, as predicted (Figure 1). Conclusion: Our screens suggest that KD point mutations may represent the primary mechanism of acquired TKI resistance in EBF1-PDGFRB Ph-like ALL. T681I was the most common KD point mutation in EBF1-PDGFRB upon exposure to imatinib or dasatinib. Future efforts should focus on targeting the T681I gatekeeper mutation with ponatinib or alternative agents for relapsed Ph-like ALL patients harboring these mutations. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 2. Figure 2. Disclosures Smith: Astellas: Research Funding; Plexxikon: Research Funding. Mullighan:Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.

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Natural Genetic Exchanges between Vaccine and Wild Poliovirus Strains in Humans

Journal of Virology ◽

10.1128/jvi.74.18.8434-8443.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8434-8443 ◽

Cited By ~ 146

Author(s):

Sophie Guillot ◽

Valérie Caro ◽

Nancy Cuervo ◽

Ekaterina Korotkova ◽

Mariana Combiescu ◽

...

Keyword(s):

Paralytic Poliomyelitis ◽

Nucleotide Sequencing ◽

Wild Type ◽

Coding Region ◽

Wild Poliovirus ◽

Type 3 ◽

Recombinant Genome ◽

Natural Means

ABSTRACT In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3′ moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

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Genetic mutations influencing ovulation rate in sheep

Reproduction Fertility and Development ◽

10.1071/rd01078 ◽

2001 ◽

Vol 13 (8) ◽

pp. 549 ◽

Cited By ~ 41

Author(s):

K. P. McNatty ◽

J. L. Juengel ◽

T. Wilson ◽

S. M. Galloway ◽

G. H. Davis

Keyword(s):

Growth Factor ◽

Point Mutation ◽

Transforming Growth Factor ◽

Somatic Cells ◽

Follicular Development ◽

Experimental Models ◽

Ovulation Rate ◽

Corpora Lutea ◽

Coding Region ◽

Primary Stage

Ovulation rate in mammals is determined by a complex exchange of endocrine signals between the pituitary gland and the ovary, and by paracrine signals within ovarian follicles between the oocyte and its adjacent somatic cells. One approach to identifying factors regulating ovulation rate is to find mutations that influence the target phenotype and, in this context, sheep are proving to be remarkable experimental models. Recently, in three sheep families, namely Inverdale, Hanna and Booroola, the inherited mutation was mapped to a specific region of the sheep X chromosome (Inverdale, Hanna) or sheep chromosome 6 (Booroola) and in each, a point mutation was identified in genes from the bone morphogenetic protein (BMP) relatives of the transforming growth factor &lsquor; superfamily or their receptors. In Inverdale (I) and Hanna (H) sheep, separate point mutations were identified in the BMP15 gene corresponding to sites in the mature peptide coding region of the BMP15 growth factor (also known as growth differentiation factor 9B; GDF9B). Expression of the BMP15 gene was located exclusively in oocytes from the primary stage of follicular growth. There is a complete block of normal follicular development in females carrying two copies of the Inverdale mutation (II), two copies of the Hanna mutation (HH), or one copy of each mutation (HI). Increased ovulation rates are found in females with only one copy of either mutation (I+ or H+). In Booroola sheep, a point mutation was identified in the highly conserved intracellular serine threonine kinase signalling domain of the BMP-1B receptor. Within the ovary, this gene is expressed in oocytes in primordial and pre-antral follicles and in granulosa cells from the primary stage of growth as well as in corpora lutea. The effect of the Booroola mutation is additive for ovulation rate: animals with one copy of the mutation have an ovulation rate of 3 or 4, whereas those with two copies have an ovulation rate of between 5 and 14. Physiological studies of the above mutations demonstrate that the oocyte plays an active role with respect to its adjacent somatic cells during follicular development and support the hypothesis that the oocyte has a significant influence on the number of follicles that proceed to ovulation.

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Macaca fascicularis papillomavirus type 1: a non-human primate betapapillomavirus causing rapidly progressive hand and foot papillomatosis

Journal of General Virology ◽

10.1099/vir.0.006544-0 ◽

2009 ◽

Vol 90 (4) ◽

pp. 987-994 ◽

Cited By ~ 15

Author(s):

Joongho Joh ◽

Kelly Hopper ◽

Koenraad Van Doorslaer ◽

John P. Sundberg ◽

Alfred B. Jenson ◽

...

Keyword(s):

Genomic Dna ◽

Macaca Fascicularis ◽

Cynomolgus Macaque ◽

Pcr Amplification ◽

Open Reading Frames ◽

Coding Region ◽

Primate Model ◽

Hands And Feet ◽

Human Primate

Papillomaviruses (PVs) are a group of small, non-enveloped DNA viruses that cause mucosal or cutaneous neoplasia in a variety of animals. Whilst most papillomas will regress spontaneously, some may persist or undergo malignant transformation. In this study, aggressive, persistent and extensive warts were observed on the hands and feet of a cynomolgus macaque (Macaca fascicularis). The presence of PV in the wart biopsies was identified by immunohistochemistry and PCR amplification of PV DNA. The genomic DNA of this PV was cloned and sequenced, and the PV was designated M. fascicularis papillomavirus type 1 (MfPV-1). Its genome was 7588 bp in length and the organization of its putative open reading frames (E1, E2, E6, E7, L1, L2 and E4) was similar to that of other PVs. MfPV-1 had a short non-coding region (NCR) of 412 bp. Molecular analysis of MfPV-1 genomic DNA classified it into the genus Betapapillomavirus, to which all epidermodysplasia verruciformis (EV)-type PVs belong. Diseases caused by PVs of the genus Betapapillomavirus are usually associated with natural or iatrogenic immunosuppression. The genomic characterization performed in this study showed that MfPV-1 clustered within the genus Betapapillomavirus and also contained EV-type-specific motifs in its NCR. Further characterization of this virus and its host interactions may allow us to develop a non-human primate model for human betapapillomaviruses, a genus populated by human PV types causing EV.

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Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651583 ◽

1993 ◽

Vol 69 (03) ◽

pp. 217-220 ◽

Cited By ~ 12

Author(s):

Jonathan B Rosenberg ◽

Peter J Newman ◽

Michael W Mosesson ◽

Marie-Claude Guillin ◽

David L Amrani

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Point Mutation ◽

Genomic Dna ◽

Comparative Sequence Analysis ◽

Chain Gene ◽

Molecular Defect ◽

Nucleotide Position ◽

Normal Individuals ◽

Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

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