Citation
The Functional Characterization of Bx34, a Drosophila homologue of Tpr

Material Information

Title:
The Functional Characterization of Bx34, a Drosophila homologue of Tpr
Creator:
Havrilesky, Michael
Zhou, Lei ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

Subjects

Genre:
serial ( sobekcm )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.

Downloads

This item has the following downloads:


Full Text








JOurn.31 ofr in.nerr.3adua.3e -:e-earch

..Oluii 5, is.ue 2 - rio.-e l-i er 2:i::-3



The Functional Characterization of Bx34, a Drosophila homologue of Tpr

Michael Havrilesky


ABSTRACT


Preliminary functional analysis of Bx34, a previous uncharacterized Drosophila melanogaster protein with

substantial sequence and amino acid similarity to human nuclear pore complex protein Tpr, is reviewed here.

A common genetic technique of transposable p-insertion was utilized to study the defects resulting from a mutation

in this area of the Drosophila genome. Additionally, various immunocytochemical techniques were used to

visualize these extensive malformations in neuronal development, predominantly found to be in the CNS and PNS

of middle stage, recessively lethal mutant embryos. Furthermore, this paper attempts to attribute the

abnormalities to the mutated gene product through a rescue of lethality via a complete Bx34 reversion by

jumping out the p-element. And furthermore, the data presented begins to shed light on the functional significance

of Bx34, which subsequently leads to speculation of human Tpr functional similarity as a result of primary

and secondary structural parallels. Information resulting from these genes may provide possible

biomedical implications into phamacodynamics and cancer research.



INTRODUCTION


As the organelle housing the genome, the nucleus plays a fundamental role in the operation of the cell. A

large number of essential and complex functions occur there, including DNA packaging and replication,

RNA transcription, and mRNA transport (Demerec, 1965). The number and complexity of these tasks has long led

to suggestions of internal nuclear structures that organize and facilitate these functions. Recent evidence points to

a membrane bound Nuclear Pore Complex containing cytoplasmic filaments projecting from its core (Davis et

al., 1995). Transport substrates dock to these filaments and translocate through the transporter on their way in

and out of the nucleus. A variety of immunological, biochemical and genetic techniques have been

successfully employed in the past few years to identify NPC component proteins (nucleoporins) Berezny et al

(1995), Bangs et al, 1996).



While it is clear that the NPC is a major regulator of nucleocytoplasmic transport, the knowledge of how this

essential superstructure is connected both structurally and functionally to the nuclear interior is still

extremely limited. Recently, the investigation of the molecular basis of such connections has received great





impetus from the study of vertebrate Tpr (translocated promoter region) and of the Tpr homologous

Drosophila melanogaster protein Bx34 (Byrd et al, 1994). Recent literature has pointed to this protein being

found entirely in the nucleus and is barred from the chromosomes and the nucleous of the interior and usually

is expressed between these structures and the nuclear periphery (Zimowska et al, 1997). Furthermore, Bx34

shows moderate sequence similarity over its entire length to the mammalian NPC protein Tpr from 28% amino

acid identity and 50% similarity (Byrd et al. 1994; Zimowska et al. 1997). The Bx34 is found in the 48C region of

the 2R chromosome and it's cDNA sequence predicts two distinct structural domains: a large N-terminal domain

(180 kD) strongly predicted to form a coiled-coil and an acidic C-terminus (82 KD) predicted to be

unstructured (Mitchell, et al, (1992). It is this data indicated that theBx34 coiled-coil region interacts with the

nuclear interior as well as the nuclear pore complex. This data compounded with its high level of sequence

homology with the mammalian Tpr, a known nuclear pore complex protein has lead to speculation hinting at

the functional relation of Bx34 to that of the mammalian Tpr as playing a role in nucleo-cytoplasmic transport but

no known function of the gene has been studied as of currently (Zimowska et al. 1997). The applicational

significance Tpr possesses could range from drug design to oncological relevance.



In lieu of recent literature reporting the structural and molecular significance of Bx34 and its tremendous degree

of homology to the mammalian NPC protein Tpr, the next feasible step is utilizing the determined genomic location

of Bx34 in a smaller eurkaryote for exploration on the functions of this protein. Drosophila serves as an ideal

model to perform genetic characterizations for insight into its function. The goal of this paper has been to

provide functional implications through biochemical analysis of a recessive Bx34 mutant carrying a p-

element insertion in the Bx34 allele of Drosophila embryos to begin to illuminate the role of this protein.

Additionally, subsequent reversion of the gene product through cross-induced p-element excision allowed creation

of a nonlethal population homozygous for Bx34 gene.



METHODS AND MATERIALS


Drosophila stocks


An overview of the series of crosses carry out for this project as illustrated in Figure 1. The

schematic displays the order of crosses initiated to revert the Bx34 recessively lethal mutant

obtained from Bloomington Drosophila Stock Center. Due to space restrictions, a more

detailed description of the cross used is unavailable, however all information on the genotypes can

be obtained at www.flybase.org. The BDSC stock number utilized was known as 10537 and this

strain carries a transposable element called a p-insertion of 48 kb engineered into the first intron of

the Bx34 transcript located at 48C5- 6 on the 2R chromosome. Effectively disrupting all viability of

the gene product. The approach used to revert the mutation was through excision of the p-

element through the introduction of a stock containing transposase labeled G90: Transposase is

only transcribed in the germ cells thus, influencing the excision event. The primary cross utilized in

this project was initiated through cross of the p insertion stock 10537: y1 w 67c23; pw+mCC=lacwLl





(2) k03905 k03905/ CyO with the transposase containing stock (G90) of w;Dr/TmS SB P{ry(+)delta 2-

3} 99B. The 10537 carried the mini-white marker for insertion verification phenotype, as well as a

lacZ transcript for biochemical analysis. Once the excision was determined to have occurred

through screening of the presence of various markers, the G116 or B-1672 stock number at BDSC,

CyO balancer stock was introduced to stablize the genetic excision event through suppression

of recombination. This Curly 0 balancer was utilized based on its high viability when used to

stabilize genetic manipulations on the second chromosome.





Overview of Drosophila Reversion Cross

Fe ylw 64;� placV) 1(2) kOW,'5 "*'* Qvo X ,'rTS SB Pr'3 +.-iia 2-3) 99B
7'-

F) w67c23: FIK IS'/ 1(2) 1 / +, TIMS (02-31 + X wilfl J rv4.Sojcf PfrI+r' 2/=enlJivgfenIIJ


F2 ii ''3; pictl.) r(2) k030S ' /0c,; TMS (02-3V + X wflllSI.nocf[&xcJ/)O.Pro.f+i 2j=ennlwgenl IJ


F3 wiv (x)pfaw,)W 1(2) koi39O S^S m O X w[ll IS] nD.-[c.otiCO P(ry[+t- )=cn,-]l,..[nil I]


F4 wL1, .pj1air I(2)kOMSO"S5/ cy'O X ^, 2r ..;i;',"i-tgwiusmsC O


FS L.f.7: . rfj p/;a[;.i',: l,. t0: A ,5o. 1 , I . 2"., Px. ilI'ni'd w63. '




Figurel. A stepwise view of the main cross for the reversion of the 10537 mutation. All genotype

mutations are displayed with their respective www.flybase.org abbreviations. The final revertant is

seen as male or female viable homozygous for the excision of the insertion labeled (ex). Arrows mark

as male or female viable homozygous for the excision of the insertion labeled (ex).




Color Balancer

Additionally, a small cross was carried out with the original stock of 10537/CyO to obtain a

biochemical way to accurately distinguish the genotype of embryos once antibody stained. A 10537

color balancer was created, so-named because of the lacZ marker fused with the Drosophila

wingless gene balanced with CyO, so as to acquire a stock that would be able to distinguish

the homozygote 10537, which contains two copies of the insertion, from the heterozygote when

stained with the monoclonal antibody beta-Galactosase. Effectively, the embryos utilized from this

cross displayed deficient patterns based upon the their genotypes, when stained using B-gal. This

stain used in conjunction with other monoclonal antibodies served as an obvious marker for analysis

of data.







Immunocytochemistry

Drosophila melanogaster embryos from stages 0-15 of development were collected and divitellinized

with 40% PEMS, 5% Formaldehyde and 55% Heptane mix and stored at -20C in methanol. Using

an index of antibodies at the University of California at Berkeley, a list of monoclonal antibodies

were chosen for nervous system staining of the fixed embryos for further analysis of the excision

event and mutant characterization with all dilutions using a 1XPBT + 5% horse serum stock. Anit-

beta-galactosidase was used to stain for presence of lac-z operon, most effectively at 1:4000

dilution. Elav-9F8A9 stained for the drosophila protein elav. It is a marker for most cells in the

central and peripheral nervous system, at all stages of development, once they have differentiated into

a neuron. After numerous trials the dilution of 1:10 antibody to 1XPBTH solution proved to stain

most profoundly with low traces of background staining. The third monoclonal antibody Bpl02 anti-

CNS axons staining strongly for the axons of the CNS with virtually undetectable staining of neuron

cell bodies, the PNS, or any other tissues of the embryo at a 1:20 dilution. All staining procedures

utilized a secondary antibody of Biotinylated horse anti-mouse 1:400 dilution with a tertiary antibody

of 1:1:100 diluted AB complex provided by Vector laboratories.



Additionally, the in situ hybridization was conducted with anti-Tpr probe using Dig RNA labeling

obtained from Boehringer M and according to the protocol provided by the manufacturer.



RESULTS



Preliminary testing of in situ hybridization with anti-TPR probe conducted with the 10537 color

balancer fly stock embryos from various age groups of 0-18 hours gave sufficient evidence of high

levels of expression in the CNS and PNS development of early stage embryos. The in situ procedure

was conducted in conjunction with anti-Beta Galatosase staining to use as a distinct marker of the

lethal homozygous progeny. It was the homozygous embryos that sparked interest particularly

because it was these that displayed the aforementioned abnormalities in development; they

are distinguished from the heterozygous by lack of identical ectodermal segments. These patterns

are evident in figure 2, which displays a sagittal and dorsal view, respectively of two stage 10-

12 embryos, which vividly show these ectodermal bands. This evidence hinted at possible

functional significance of the Bx34 in neuronal development.





















Figure 2. Monoclonal antibogy staining with B-gal of embryo created from the 10537 color balancer

stock. Stage 6-7 embryos display the early ectodermal lacZ markers. Dorsal view is seen left with the

right figure shown from a sagittal view.




This data being said, the next phase of the research naturally, focused on determining the precise

region of the affected neural development the p-inserted Bx34 mutants. After a general indication

of expression of the transcript in the developing nervous system a more focused approach

toward proving Bx34 was required for normal neural development was executed. Monoclonal

antibody staining recognizing drosphila proteins expressed during CNS and PNS development

was utilized. Elav-9F8A9 and BP102 anti-CNS proved to be most significant in their effectiveness

for displaying the profound defects in developing embryos. Figure 3 displays a comparison of various

Elav stained 10537 homozygous mutants with that of viable Oregon R wild type embryos all

at approximately stage 12 of development. The upper left displays a sagittal view of a wt embryo

with normal Cns development and below is a dorsal view of the same embryo. The left-hand

column illustrates the same stage embryos displaying the significant neuronal defects of the mutation

at the Bx34 allele. Dramatic nervous system damage is evididant in both the developing

neuroblasts, precursors of the central nervous system, and staining is also seen in the immature

sensory neurons that will eventually make up the peripheral nervous system. Correlations to the

nervous system damage are seen in Figure 4, as well, which illustrates the Bp-102 staining of

similar stage embryos. It was determined from figure 2, 3 and further staining data that lethality

occurs in the mutants variable on the level of Bx34 expression, however from general data few

mutants developed past stage 13.









.I.ImI~,


� IH-A^f


b)







Figure 3. Monoclonal antibody staining with Elav 9F8A9.. a) and c). Slightly different dorsal angles of
Oregon R wildtype. b) and d). Mutant 10537 color balancer embryos shown both dorsally. All embroys
shown are approximately at stage 10-11.











Figure 4. A sagittal view of BP102 stained embryos stages 7-8. Left picture homozygous lethal
embryo.



A rescue from the lethality of the nervous system defect was accomplished through the list of
crosses illustrated in the methods and materials section and seen in figure 1 via the successful
excision of the p-insertion. In other words, the crosses were considered a success if the final screening
of progeny showed phenotypic markers indicating a viable Bx34 transcript having total precise
excision of the p-element insert. Thus, achieving total reversion of the homozygous lethality.
Upon complete reversion of the homozygous lethal strain to the viable Bx34 allele, normal
embryonic development occurred. This is apparent from the screening of the final progeny for
correct phenotype. The suspected genotypic ratios should follow the classical Mendalian genetic
ratios. In Table 1,the progeny are scored based on whether they show the revertant phenotype or are
of phenotype showing the excision event did not occur precisely. The final scores of the revertant
cross are shown here in Table 1. As the numbers of the vials indicated, not all of the vials
created produced a strain of revert ant flies with the correct phenotypes, this could be from the a
number of reasons from excision not occurring at all or possibly due to imprecise excision of the
p-insertion which occurs usually at the frequency of 5% to 95% precise excision in nature. Although
the percent of recovery of all crosses does not follow this ratio, a number of other factors could
explain this from the level of viability of the original fly stock or the environmentally factors such
as fecundity of the flies utilized.








Table 1

Final Phenotypic Ratios of Reversion Cross


Mutant Phenotype


Revertant WT Phenotype


11A-2 5\12 7\13 17\20

11A-3 1\0 3\2 1 \5

16B-2 8\4 18\15 12\33

10A-1 0\0 4\7 4\7

10D-1 12\14 11\16 26\27


Table 1: The above table displays the final progenial scores of the crosses made to successfully excise the p-insertion from

the genome thus creating a viable Bx34 gene. The numbers shown are in ratio of males to females after a period of two weeks

of screening.



DISCUSSION



The results of this paper clearly display that the dramatic developmental defect in both the CNS and PNS of

mid-stage Drosophila embryos is a direct correlation with the disruption of the Bx34 transcript and that once

this alteration is removed from the genome these abnormalities cease to cause recessive lethality and a

viable homozygous fly remains. Further research is needed to draw exact comparison to human Tpr, however, due

to the extensive biochemical homology between this and Bx34, a strong case has been made for a possible

similar function. Nonetheless, the data reported shows promising evidence for future scientific studys.



Finally, I must thank some people whom without this paper would have not been possible. Gina Chan and Dr.

Rong Yuan of the Department of Molecular Biology and Genetics at the University of Florida for their advice

and support also, my mentor Dr. Lei Zhou who donating priceless knowledge, expertise, and time to aid in

this project's success.


REFERENCES



Bangs, P.L., Sparks C. A., Odgren, P. R. and Fey, E. G. (1996) "Product of the onco-activiating gene Tpr is

a phosphorylated protein of the nuclear pore complex." J. Cell. Biochem. 61, 48-60.



Berezney, Ronald, Mortillaro, Michael, Ma, Hang, Wei Xiangyum, (1995). "The nuclear matrix: a structural milieu

for genomic function." Int. rev.of Cytol. 162A, 1-65.


Tube #


Totals






Byrd, D. A., Sweet, D.J., Pante, N., Knostantiniov, K. N., Guan, T., Saphire, A.C. (1994). "Tpr, a large coiled

coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the

cytoplasmic surface of the nuclear pore complex."J. Cell Biol. 127, 1515-1526.



Davis, Laura. (1995) "The nuclear pore complex." Annu. Rev. Biochem. 64, 865-896.



Demerec, M. (1965) Biology of Drosophila, pp. 344-350. Hafner Publishing, New York.



Mitchell, P. J. and Cooper, C. S. (1992). "The Human tpr gene encodes a protein of 2094 amino acids that

has extensive coiled-coil regions and an acidic C-terminal domain. " Oncogene 7, 2329-2333.



Zimowska, Grazyna, Aris, J. (1997) "A Drosophila Tpr protein homolog is localized both in the

extrachromsosomal channel network and to nuclear pore complexes." Jour. Of Cell Sci. 110; 927-944


--top--



Back to the Journal of Undergraduate Research


College of Liberal Arts and Sciences I University Scholars Program I University of Florida I


� University of Florida, Gainesville, FL 32611; (352) 846-2032.


S UNIVERSITY of
UF FLORIDA