Course Syllabi And Assessment Information

Cathedral City High School Bands

Symphonic Band & Concert Band Performance & Course Information.

Matthew Howe, Cathedral City High School Director Of Bands

Adjunct Instructor – College of the Desert

“Excellence Is Our Minimum Standard”

Ensemble Goal  -”Developing The Artist”:

“The artist has all the skills of the player and the musician, but the artist is also a creator. The artist comes to every rehearsal prepared in every way, and leaves the rehearsal with new goals. The artist loves great music making and loves to bring expression and inspiration to the performance. The artist has imagination this is fueled by opportunity. That opportunity might come in a solo passage or in an approach to style that amplifies the intent of the piece. The artist is a collaborator with the other members of the ensemble, with the conductor, and the composer. The artist is intuitive and original, but only uses those skills in pursuit of the most beautiful performance possible. The artist evaluates whether a piece of music is good or not by how it is composed and what it expresses. The artist has the potential to evaluate the listener’s perception of an average piece through an extraordinary performance. The artist loves music because music fuels his/her soul”

- Allan McMurray

Description:

Great artists prepare themselves for rehearsal through daily preparation. In order to assess each student in this preparation, mini-juries are held bi-weekly.

“Mini – Juries” are playing exams. Symphonic Band “mini juries” will occur twice per month as scheduled on the band calendar by section/instrument grouping. Concert Band “mini juries” will occur weekly (typically on Fridays).  The purpose of each “mini-jury” will be to assess the performers level of preparedness in completing technique and literature on an individual basis. The grading policy for both Concert Band and Symphonic Band are listed below. The literature for each mini-jury for Symphonic Band follows, Concert Band information will be assigned in class on Mondays.

Each student will be assigned a “mini-jury” performance time by section. Symphonic Band students are responsible for coming in and completing the jury on their requisite assessment day (Concert Band “mini juries” will be assigned weekly and performed in class). Students are encouraged to use the Cathedral City High School Day Planner for accurate preparation planning and scheduling. Please note that this planner is provided free of charge by CCHS. All mini jury times and dates can be found on the “News/Calendar” link of the CCHS Band website (www.cchsbands.org). Please note that for Symphonic Band students, placement in the performing ensemble is decided by the scoring the takes places as a result of a student’s mini-jury performance. LOW SCORING STUDENTS WILL NOT BE ALLOWED TO PERFORM IN THE CONCERT WITHIN THAT TEST CYCLE DUE TO A LACK OF PROFICIENCY IN INDIVIDUAL ASSESSMENT. Students NOT enrolled in the scheduled symphonic band course will take an additional “supplemental exams” monthly as scheduled by the Director of Bands.

Note: Mini-juries are “tiered” exams. Specifically, the technique portion of the exams (scales, arpeggios, chromatic triplets) count as HALF of the musician’s mini-jury score. The technique portion of the jury is the first portion of the mini-jury. Students who cannot pass the technique portion of the exam with an “A” average will not be allowed to move on to part two of the mini-jury (where the literature of the ensemble is assessed and scored). If a student does not pass the technique portion of the mini-jury, then the student will fail that particular mini-jury with a score of 50 percent or “E”.

Grades for “mini juries” will be assessed along the following rubric:

Rubric Score: Grade: Description:

25 Points             A

Well prepared. No mistakes on literature or technique requirements. Musical terms are defined exactly, students perform in time with metronome.

20 Points              B

Some mistakes in literature and technique. Some musical terms are defined. Student performs in time with metronome.

15 Points              C

Average performance. Many mistakes in performance. Musical terms are either missing exact definition, or are not defined. Student does not perform in time with metronome.

10 Points              D

Too many mistakes in performance. No terms are defined. Student not in time with metronome.

“Full Juries”(Symphonic Band Students Only)

Being a great artist involves the serious study of the major etudes and concertos for each musicians instrument. As a part of the course students will end each semester by performing full juries. Full Juries are the major culminating activity of the fall and spring concert season. Juries are a standard performance requirement at a University- level music school. Students in the CCHS Symphonic Band will be assigned an etude, concerto, or solo at the outset of the concert season, and will prepare the work along with the compulsory technique exercises in their private lessons. These items will then be presented by the student in a recital-format before a “jury” of CCHS Band staff and music coaches, with principal chair students performing accompanied juries with a pianist. Full Juries are weighted higher than band concerts, and mini-juries. The total point value for Full Juries is 1000 points. Poor performance at at full jury will result in removal from the ensemble.

Performance Forums:

Pre-jury performance forums will be scheduled throughout the fall in order to assist students in preparing for their full jury. Students will be encouraged to partake in these forums in order to make their jury performance as musical as possible.

Performance Requirements:

In order to perform in concert, a Symphonic Band/Concert Band student must not have more than one “D” on any performance exams. Students who do not attend their section’s assessment day due to student unpreparedness, forgetfulness, etc., will automatically fail their “mini-jury”. Students who do not perform on a concert due to the aforementioned policy will have their course grade lowered two full letter grades. Additionally, as this is a performance based course, students who miss concerts due to ANY other reason outside of illness (with a doctor’s note), will be dropped out of the course at the semester. Concerts are weighted at 200 points per event. Symphonic Band students with more than one “D”  in one semester will be dropped from the course at the semester break.

Course Materials:

All musicians who are members of the Symphonic and Concert Bands need to have  the following for optimal course success:

  1. Reed players must have a swab for their instruments (silk swabs for clarinets), and a reed holder that holds a minimum of four reeds.
  2. Reed Players: FOUR fully-functioning reeds at all times.
  3. Brass Players: Necessary Oils/Valve And Slide Cream.
  4. Fully Functioning Metronome and Tuner.
  5. Pencils on stands.

Note: These items may be purchased at local music shoppes, and through the internet. Please see the Director of Bands to get further information.

A Word About Music….

Great artists come to EVERY rehearsal prepared for the rehearsal to come, which includes the preparation of all materials that are required for the course. Lost, or forgotten music is unacceptable in an ensemble with the pedigree of the CCHS Bands. Students who are not musically prepared at rehearsal will have their grade lowered by a quarter grade (an A becomes an A-). Leaving music at home, or in the car, or anywhere else except having it at the rehearsal setting is inexcusable.

Attendance At Rehearsals:

Great artists are prepared and present at all scheduled rehearsals and sectionals. Students are expected to attend all rehearsals as scheduled. The CCHS Bands website contains the CCHS Bands calendar, which holds all of the information needed in order to successfully manage the requisite course rehearsal dates. Saturday rehearsals are required for Symphonic Band Students, and each Saturday rehearsal is weighted as a test grade. Please note missing more than one Saturday rehearsal will equate to a student not performing on the forthcoming concert. Students who do not perform on a concert due to the aforementioned policy will have their course grade lowered two full letter grades. Additionally, as this is a performance based course, students who miss concerts due to ANY other reason outside of illness, will be dropped out of the course at the semester. There are NO excused absences, and after 5 absences for any reason, a student will have their grade lowered by one quarter grade.

There are many prerequisites for participation in the CCHS Bands but among the most important requirement is the personal commitment to be prepared for rehearsals.  The purpose of our rehearsals is to learn the other musicians’ parts – not to learn your own partLikewise the other musicians cannot learn your part unless you are prepared and in attendance. Therefore your attendance is mandatory at all rehearsals and individual preparation is required. Musical honesty and integrity in concert grows out of our commitment to being prepared for rehearsals.  Every rehearsal is a performance leading up to the culminating experience of the concert.

Individual Preparation and Practice

“Great artists are great artists because they enjoy practice as much as performance.  That is because their practices (or repetitions) are always performances.  Repetition in rehearsal is both repetition and deepening.  One must experience focused repetition in order to discover the mysteries of the art.  One must experience repetition in order to explore the beauties of the piece.  One must repeat in rehearsal in order to learn a fundamental human condition: commitment.”

-James Jordan

The description of practicing quoted above is significant in that it describes the essence of what is required to be an excellent performing musician.  The degree to which we commit to this attitude will be reflected in the quality of our work as individuals and as an ensemble.  Practice often and practice with specific goals in mind.  Be sure to practice those skills you need the most work on work on the music you will be accountable for in upcoming rehearsals.

Band Stage Productions – Winter Semester:

As Symphonic Band and Concert Band are year-long courses, Symphonic Band students must elect Band Stage Productions in addition to the regular Symphonic Band class in the second semester. This component of the course will meet from 3 pm – 5 pm (estimated) in the winter semester. Instructional coaches will teach sectionals and rehearsals in order to prepare the students for winter festival and concerts. Please note that this is a graded class that occurs in the seventh period of the school day.

Private Lessons:

Great artists seek out and learn from qualified pedagogues in their field in order to deepen and broaden their skill set and musicianship. Students enrolled in the CCHS Bands will have the opportunity to study privately. This opportunity is a way to further the performance goals of both the student, and the overall ensemble. Information regarding private lessons will be discussed at the outset of the concert season. Note: CCHS Symphonic Band Students will need to take two private lessons per month as a compulsory assignment of the course. Private lessons are paid by the individual student and can be financed through the many fundraisers scheduled throughout the school year by the CCHS Band Boosters. Please note that lesson attendance is mandatory for successful course completion, and canceling lessons in less that 24 hours will result in financial penalties to families, as many of our private teachers drive a considerable distance to teach here at CCHS. Please note that missing private lessons will equate to students NOT being allowed to perform in concert, or maintaining a position in the course.

All Coachella Honor Band & Solo/Ensemble:

All members of the CCHS Symphony Band MUST complete BOTH of the following solo/ensemble requirements

  1. Audition For All Desert Honor Band in March, 2017
  2. Perform in one Solo/Ensemble Festival in February or April of 2017

Honor Band Auditions:

All students who sit as principal chair players must audition for two of the following honor ensembles:

SCSBOA All Southern Honor Band

CMEA All State Honor Band

RCC John Philip Sousa Honor Band

CSU LA First Chair Honor Band

SCSBOA Solo/Ensemble Festival

All-Coachella Honor Band

All musicians who are not principal chair players may audition for these honor groups as well. Please be sure to calendar all honor group rehearsals and events so as to avoid conflicts with CCHS Band events.

Artists Don’t…..

Lose their music.

Arrive late to rehearsal.

Neglect their individual warmup prior full rehearsal beginning.

Forget or neglect their pencil at rehearsal.

Neglect the study of the entirety of a piece by using all resources available to them to

understand everything they can about a piece they are performing.

Make excuses for why they cannot attend rehearsals/lessons.

——————————————————————–

Cathedral City High School Bands

Jazz Band & Percussion Ensemble Performance “Mini – Juries” And Requisite Course Information. Band

Description: “Mini-Juries”:

“Mini – Juries” are playing exams. All Jazz Band and Percussion Ensemble “mini juries” will occur weekly.   The purpose of each “mini-jury” will be to assess the performers level of preparedness in completing technique and literature.

Please note all “mini juries” will be performed in class. Students are encouraged to use the Cathedral City High School Day Planner for accurate preparation planning and scheduling. Please note that this planner is provided free of charge by CCHS. All mini jury times and dates can be found on the “News/Calendar” link of the CCHS Band website (www.cchsbands.org).

Grades for “mini juries” will be assessed along the following rubric:

25 Points      A

Well prepared. No mistakes on literature or technique requirements. Musical terms are defined exactly, students perform in time with metronome/rhythm section.

20 Points      B

Some mistakes in literature and technique. Some musical terms are defined. Student performs in time with metronome/rhythm section.

15 Points      C

Average performance. Many mistakes in performance. Musical terms are either missing exact definition, or are not defined. Student does not perform in time with metronome/rhythm section.

10 Points      D

Too many mistakes in performance. No terms are defined. Student not in time with metronome/rhythm section.

Jazz Band/Percussion Ensemble “Gigs”:

Throughout the year, both Jazz Band and Percussion Ensemble will have scheduled performances outside of the regular school day. These events will be calendared at least two weeks in advance, and will be weighted as any other performance. Absence from these performances will result in a two letter grade deduction in a student’s grade.

Performance Requirements:

In order to perform in concert, a student must not have more than one “D” on any mini-juries. Students who do not attend their section’s assessment day will automatically fail their “mini-jury” (only excused with a SIGNED doctor’s note). Students who do not perform on a concert due to the aforementioned policy will have their course grade lowered two full letter grades. Additionally, as this is a performance based course, students who miss concerts due to ANY other reason outside of illness, will be dropped out of the course at the semester. Concerts are weighted at 200 points per event. Band students with more than one “D”  in one semester will be dropped from the course at the semester break.

Course Materials:

All musicians who are members of the Jazz Band and Percussion Ensemble need to have  the following for optimal course success:

  1. A fully functioning metronome.
  2. A music dictionary: Cited online at the CCHS Bands Website.
  3. Reed players must have a swab for their instruments (silk swabs for clarinets), and a reed holder that holds a minimum of four reeds.
  4. Reed Players: FOUR fully-functioning reeds at all times.
  5. Brass Players: Necessary Oils/Valve And Slide Cream.

Note: These items may be purchased at local music shoppes, and through the internet. Please see the Director of Bands to get further information.

Attendance At Rehearsals:

Students are expected to attend all rehearsals as scheduled. The CCHS Bands website contains the CCHS Bands calendar, which holds all of the information needed in order to successfully manage the requisite course rehearsal dates. Saturday rehearsals may be required for Percussion Ensemble and Jazz Band Students, and each Saturday rehearsal is weighted as a test grade. Percussion Ensemble students performing with the Symphonic Band or Concert Band are subject to their rehearsal schedule as well. Please note missing more than one Saturday rehearsal will equate to a student not performing on the forthcoming concert. Students who do not perform on a concert due to the aforementioned policy will have their course grade lowered two full letter grades. Additionally, as this is a performance based course, students who miss concerts due to ANY other reason outside of illness, will be dropped out of the course at the semester.

There are many prerequisites for participation in the CCHS Bands but among the most important requirement is the personal commitment to be prepared for rehearsals.  The purpose of our rehearsals is to learn the other musicians’ parts – not to learn your own part.  Likewise the other musicians cannot learn your part unless you are prepared and in attendance. Therefore your attendance is mandatory at all rehearsals and individual preparation is required. Musical honesty and integrity in concert grows out of our commitment to being prepared for rehearsals.  Every rehearsal is a performance leading up to the culminating experience of the concert.

Individual Preparation and Practice:

“Great artists are great artists because they enjoy practice as much as performance.  That is because their practices (or repetitions) are always performances.  Repetition in rehearsal is both repetition and deepening.  One must experience focused repetition in order to discover the mysteries of the art.  One must experience repetition in order to explore the beauties of the piece.  One must repeat in rehearsal in order to learn a fundamental human condition: commitment.”

James Jordan

The description of practicing quoted above is significant in that it describes the essence of what is required to be an excellent performing musician.  The degree to which we commit to this attitude will be reflected in the quality of our work as individuals and as an ensemble.  Practice often and practice with specific goals in mind.  Be sure to practice those skills you need the most work on work on the music you will be accountable for in upcoming rehearsals.

Attendance At Rehearsals, Performances, and Jazz Gigs:

Students are expected to attend all rehearsals, Performances, and Jazz Gigs as scheduled and presented to the students. The CCHS Bands website contains the CCHS Bands calendar, which holds all of the information needed in order to successfully manage the requisite course rehearsal dates. Saturday rehearsals, performances or Jazz Gigs in the fall are required for Symphonic Band, Jazz Band, and Percussion Ensemble Students, and each Saturday rehearsal or Jazz Gig is weighted as a test grade. Please note missing more than one Saturday rehearsal or Jazz Gig will equate to a student not performing on the forthcoming concert. Students who do not perform on a concert due to the aforementioned policy will have their course grade lowered two full letter grades. Additionally, as this is a performance based course, students who miss concerts due to ANY other reason outside of illness, will be dropped out of the course at the semester.

Band Stage Productions – Winter Semester:

As all CCHS Band courses are year-long courses, percussion students performing with the CCHS Concert And Symphonic Band will elect Band Stage Productions in the second semester. This component of the course will meet from 3 pm – 5 pm (estimated) in the winter semester. Instructional coaches will teach sectionals and rehearsals in order to prepare the students for winter festival and concerts.

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Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya 

Qingyi YU1,2, Paul H MOORE3, Henrik H ALBERT3, Adrienne H K ROADER4 and Ray MING1Received 5 June 2005; Revised 27 July 2005; Accepted 3 August 2005.

Top of pageAbstractThe homologous genes FLORICAULA (FLO) in Antirrhinum and LEAFY (LFY) in Arabidopsis are known to regulate the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs Authenitc Mens C.J.Mosley jersey of the two woody tree species: California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa). Despite the high sequence similarity within two conserved regions, the N terminal proline rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional Authenitc White Dan Orlovsky jersey LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there Authenitc Youth C.J.Mosley jersey is only one copy Elite Youth C.J.Mosley jersey of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia, but it is expressed at a high level in Authenitc Barry Sanders jersey the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya.

Keywords: divergence, floral meristem identity gene, flower development, gene expression, molecular phylogeny

Top of pageINTRODUCTIONFlower meristem identity genes control the developmental transition of plants from the vegetative to reproductive phase. Several flower meristem identity genes have been cloned from Arabidopsis; among them, LEAFY (LFY) is one of the earliest flower meristem identity expressed genes 1, 2. LFY encodes a plant specific transcription regulator 3, 4 and plays a major role in flower initiation 1, 2. LFY transcripts are expressed at low levels during the vegetative phase, which are increased upon the transition from the vegetative to reproductive phase 5. Mutations in LFY lead to the conversion of the flower meristems into inflorescence meristems and result in the replacement of flowers by lateral shoots 6, 7, 8, 9. Constitutive expression of LFY promotes reproductive transition and results in early flowering 10, 11. In the complex flower development pathway, LFY is centrally located and acts as a link between the upstream flower timing genes and downstream floral homeotic genes 5. LFY integrates environmental and endogenous signals to control flower timing 12. It has also been shown that LFY activity is required for the activation of all the three classes of floral organ identity genes 4, 13, 14, 15.

Recent studies showed that the complex regulatory network of flower development is largely conserved across distantly related plants, especially among dicotyledonous plant species 16. LFY homologs have been cloned from several species of gymnosperms and angiosperms, including both monocots and dicots 9, 17, 18, 19, 20, 21, 22, 23, 24. Their functions are highly conserved with slight variations among different species. Constitutive expression of LFY promoted flower initiation not only in Arabidopsis 11, but also in heterologous species, such as tobacco 25, aspen 11, rice 21 and citrus 26. Ectopic expression of LFY homologous genes from other plant species in the Arabidopsis lfy mutant can partially or fully complement the lfy mutation 18, 27.

Papaya (Carica papaya L.) is a fruit crop grown widely in tropical and subtropical lowland regions. It is a dicotyledonous, diploid plant species and relatively close to the fully sequenced model plant Arabidopsis from the point of view of family level 28. Papaya is one of the few plant species that produces three sex forms, male, female and hermaphrodite, and provides a unique opportunity to examine the applicability of the floral development pathway established in hermaphrodite to dioecious plants. Our previous study demonstrated that papaya has a primitive Y chromosome controlling sex determination 29. Cloning and characterization of flower development genes from papaya will help us to understand the roles of floral meristem and organ identity Authenitc George Johnson jersey genes in sex determination and differentiation in plants.

Here, we report the cloning and characterization of LFY ortholog in papaya, PFL. It shares a high similarity and identity with the LFY orthologs from other Authenitc Youth Dan Orlovsky jersey dicot species and its expression pattern is similar to that of other orthologous genes. Our results showed that PFL is a functional ortholog of the Arabidopsis LFY gene expressed in all three sex forms.

Top of pageMATERIALS AND METHODSPlant materialsThree different papaya cultivars, SunUp, Kapoho and AU9, were germinated and planted in Kunia station, Oahu. Young leaf tissues of SunUp, Kapoho and AU9 were collected for genomic DNA isolation. Hermaphrodite and female flower buds, young leaf tissues and root tissues were collected for total RNA isolation. Shoot apical meristem (SAM), floral meristem and leaf meristem tissues from SunUp and Kapoho were collected and fixed at different floral developmental stages for in situ hybridization.

cDNA library constructionTotal RNA was isolated from hermaphrodite and Elite Blue C.J.Mosley jersey female flower buds using the method described by Hall et al 30. PolyA+ RNA was isolated from the total RNA using PolyATtract mRNA Isolation System (Promega, WI, USA) according to manufacturer’s instruction. About 5g polyA+ RNA was used to synthesize double stranded cDNA using the ZAP cDNA Synthesis kit (Stratagene, CA, USA). Size selected cDNA (100 ng) was ligated with 1g EcoRI/XhoI digested Uni ZAP XR lambda vector and packaged with GigapackII packaging extract (Stratagene, CA, USA). Libraries of 8106and 3.8106 recombinants, respectively, were generated. A total of 18,432 (48384) recombinant clones for each library were picked and archived in 384 well plates containing freezing medium. High density (18,432 clones per 22.5 22.5 cm2) and low density (4,608 clones per 22.522.5 cm2) membranes were made using a Genetix Q BOT (Genetix, UK).

Southern analysisIsolation of papaya genomic DNA from leaves was based on the procedure described by Tai and Tanksley 31 with minor modifications. Three restriction enzymes, HindIII, EcoRI and XbaI were used to digest papaya genomic DNA from the three cultivars, Kapoho, SunUp and AU9. Genomic DNA samples (10 g) were incubated with 5 U of restriction enzyme/g DNA in a total volume of 50 l at 37 for 4 h. Approximately 10 g digested genomic DNA was electrophoresed through 0.8% agarose gel in 1TBE buffer. After electrophoresis, the gel was blotted onto Hybond N+ membranes (Amersham, UK) using standard methods 32. The probes were labeled by random primer labeling system (RediPrimer II, Amersham, UK). The procedure for hybridization and autoradiography were as described by Chittenden et al 33.

In situ hybridizationApices, flower meristems, and leaf meristems of papaya were harvested at different stages and fixed in FAA fixation solution (3.7% formaldehyde, 5% acetic acid, 50% ethanol). Samples were then dehydrated in increasing concentrations of ethanol and embedded in paraffin (Paraplast Plus, VWR, PA, USA). Sections (8 m thick) were cut and mounted onto ProbeOn Plus slides (Fisher Scientific, PA, USA). The amplified PFL cDNA fragment was cloned into TOPOII vector (Invitrogen, CA, USA). Antisense and sense RNA probes were generated with opposing SP6 and T7 promoters from the PFL cDNA subclone and labeled with digoxigenin (DIG) (Roche, Germany). The methods for tissue pretreatment and in situ hybridization were as described by Jackson 34.

RT PCR analysisTotal RNA was treated with RNase free DNase I (Promega, WI, USA) and quantified using a spectrophotometer (Shimadzu, Japan). About 5 g of total RNA were reverse transcribed using RETROscript Kit (Ambion, TX, USA). PCRs were performed on the cDNA using the primer pair PFL Sub10 3 (5′ CCA GAA CAT AGC CAA GGA GC) and PFL 2403 (5′ AAT GGC AAG ACG AGG ATG TG).

Real Time quantitative RT Elite Dan Orlovsky jersey PCR assaysOne primer pair (A1: 5′ ATA GTG CAG GCG CCA GTC AG; A2: 5′ GGC TCA AGC TGT TCA TCA TC) had a Tm of >55 and was designed using Clone Manager 6 (Sci Ed Central) to produce a PCR product of 223 bp. About 2 g of total RNAs were treated with RNase free DNase I (Promega, WI, USA) and reverse transcribed using TaqMan RT kit Elite Barry Sanders jersey (Applied Biosystems, CA, USA). cDNA products were diluted 5.5 fold for use in real time RT PCR amplification. Platinum Quantitative PCR SuperMix UDG (Invitrogen, CA, USA) was used for real time RT PCR amplification. Reactions Authenitc Youth George Johnson jersey were run on a DNA Engine OPTICON 2 Real Time PCR detection system (MJ research, MA, USA). Thermocycler conditions were 2 min at 50 followed by 10 s at 95 and 40 cycles of 15 s at 95 20 s at 58 and 30 s at 72 For each sample, an RNA sample without reverse transcriptase was included to control for genomic DNA contamination. All presented PCR data were generated from a minimum of three independent reactions for each biological replication. Actin gene was used as a standard to control for sample variation in the total amount of RNA in different reactions. Phylogenetic analyses were conducted using the Neighbor Joining (NJ) method as implemented by the PHYLIP program package. Pam matrix was used to generate distance. Bootstrap analysis (1,000 replicates) was performed to assess the support of individual branches.

Top of pageRESULTSCloning of PFLThe Arabidopsis LFY cDNA was used as a probe to screen the papaya BAC library. Two positive BAC clones, 42B17 and 52E1, were identified and confirmed by southern hybridization. The sizes of these positive BAC clones were estimated as 60 kb and 175 kb, respectively. The smaller BAC clone, 42B17, was digested with HindIII and shotgun subcloned into pPCR Script vector. The sub clones were screened with the LFY probe and the positive sub clone was sequenced from both ends. Direct sequencing of the BAC clone was carried out at the 3′ end of the sub clone Elite C.J.Mosley jersey where the third exon of Authenitc White C.J.Mosley jersey PFL was interrupted by a HindIII site.

A full length sequence of PFL was obtained through directly sequencing of BAC clone 42B17 via a primer walking approach. Three exons were predicted based on sequence homology and conserved splicing sites. The sizes of three exons were 429 bp, 315 bp and 357 bp, respectively. Two primers were designed at the beginning of the first predicted exon and the 3′ end Elite Kids Barry Sanders jersey of the third predicted exon. The cDNA fragment of PFL was obtained by amplification of the cDNA from papaya hermaphrodite flower buds using these two primers. This cDNA fragment was cloned into pCRII TOPO vector (Invitrogen, CA, USA) and sequenced. The sequence result matched exactly the predicted open reading frames. Comparison between PFL cDNA and genomic sequence confirmed that PFL has three exons and two introns and that the splicing sites are highly conserved among PFL, FLO, and LFY.

PFL cDNA was about 1.1 kb and predicted to encode a protein of 367 amino acids. The deduced amino acid sequence of PFL was aligned with other FLO/LFY like proteins (Fig. 1). This alignment revealed two highly conserved regions and two short variable regions. A proline rich region near the amino terminus and an acidic central region were found in the variable regions of most of angiosperm FLO/LFY like proteins. The proline rich region was not found in the papaya PFL. However, a highly acidic region was observed in PFL between glutamate Elite White Deandre Levy jersey 182 and glutamate 188 where it was preceded by a short basic region (between lysine 171 and lysine 178), which is similar to other FLO/LFY like proteins. PFL also shares with other dicot FLO/LFY like proteins a region containing leucines repeated every 7 or 8 amino acids (positions leucine 69 to leucine 99) and a region with alternating Womens C.J.Mosley jersey basic Authenitc Mens Deandre Levy jersey acidic basic stretches (positions arginine 115 to arginine 126). The leucine repeats are marked with “”. The basic and acidic regions are labeled as “+” and ” “, respectively.

Full figure and legend (372K)

The comparison of PFL proteins to other FLO/LFY like proteins showed that PFL protein is more closely related to the other dicot FLO/LFY proteins than to their monocot counterparts or to gymnosperm FLO/LFY like proteins. PFL protein shares 66% and 70% similarity with LFY and its snapdragon counterpart FLO. It shares a higher similarity with LFY homolgous proteins of two woody tree species, California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa) at 75% and 76%, respectively (Tab. 1). Only one hybridizing band was seen with Hind III digestion (Fig. 2). One strong band with some weak bands were seen with EcoRI digestion and XbaI digestion. Since the sequencing result showed there are EcoRI and XbaI restriction sites within the PFL gene and only one cDNA Elite Kids Dan Orlovsky jersey fragment was amplified from the mRNA of papaya hermaphrodite flower buds, it is likely that only one copy of PFL gene exists in the papaya genome. Elite Kids Barry Sanders jersey This same probe was also used to label the papaya BAC library (data not shown). Nine positive BAC clones were obtained. All nine positive BAC clones showed the same pattern after they were digested with HindIII and XhoI and labeled with the PFL probe. This result reinforced our conclusion that only one copy of PFL exists in the papaya genome.

Figure 2.(A) Restriction map of genomic clone of PFL of selected sites. The probe used in the Deandre Levy jersey southern hybridization was indicated in the line underneath. (B) Southern hybridization result of Kapoho (K) and SunUp (S) papaya genomic DNA digested with three different enzyme, EcoRI, HindIII, and XbaI, and labeled with PFL probe. S: size control. The same fragment with the probe was used as the size control.

Full figure and legend (47K)

A phylogenetic tree was constructed using the predicted protein sequences to determine the evolutionary relationships between PFLand other FLO/LFY like proteins (Tab. 2, Fig. 3). The phylogenetic tree shows three FLO/LFY like proteins from two different gymnosperm species, NLY, PrFLL and GinLFY, clustered together and quite separate from the proteins of the angiosperms. Five FLO/LFY like proteins from monocot species, RFL, LtLFY, ZFL1, ZFL2 and JunefLF cluster in a group separate from the dicot species. LFY, VcLFY and BOFH from three different species within Cruciferae formed a distinct cluster. Similar to this, NFL1, NFL2 and TOFL from two different species within the Solanaceae formed another sub cluster. The general phylogenetic relationships found in this tree are consistent with the currently accepted species phylogenies. The PFL protein is closely related to other FLO/LFY like proteins in dicots, but it is very different from its monocot or gymnosperm counterparts.

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