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Molecular dissection of autophagic programmed cell death

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Molecular dissection of autophagic programmed cell death Powered By Docstoc
					Serial killers and their accomplices: transcription profiling of autophagic cell death.

A genomic view of autophagic cell death gene expression.

A genome wide survey of autophagic cell death gene expression.

Serial analysis of autophagic cell death gene expression.

A genomic view of gene expression in developmentally regulated autophagic cell death.

Transcription profiling of autophagic cell death.

Molecular dissection of autophagic programmed cell death.

SAGE paper; Nature or Science

Abstract, Introduction, Sections with Subheadings, up to 6 figures or tables; 40 refs only.
# words = 4500

Abstract
        Autophagic cell death is widespread in animal development and important for ___ in
____ . It is morphologically distinct from apoptotic cell death but not well characterized at the
molecular level. During metamorphosis, Drosophila larval salivary glands undergo autophagic
cell death that is activated by an ecdysone induced transcriptional hierarchy. To identify and
analyze the genes expressed, we examined wild-type patterns of gene expression in multiple
stages of Drosophila larval salivary glands using Expressed Sequence Tags (ESTs), real-time
quantitative RT-PCR, and Serial Analysis of Gene Expression (SAGE). We identified over
1,000 genes expressed differentially prior to salivary gland death, and __ of these corresponded
to potentially novel genes not in the current Drosophila predicted gene set. Genes associated
with autophagy in other organisms (yeast, other?) were identified, but many of the genes
identified have no known function. Data mining revealed genes with similarity to mammalian
FLIP and RIP family members, a mammalian BH-3 only family member, ___, and yeast
autophagy genes. [Mutant expression analysis and in situ hybridization studies indicate that
some of the genes are likely to play a role in both autophagic and apoptotic cell death, supporting
the notion that there is substantial overlap with respect to the molecules involved in these two
types of cell death. ]


Introduction

       Programmed cell death (PCD) is a critical process by which obsolete cells or tissues are
removed during normal development of multicellular organisms. Damaged cells are also
removed by PCD, providing a crucial means of controlling diseases such as cancer and
autoimmunity (? Check auto imm refs). Cell death can be divided into several subtypes based on
morphological criteria (ref). Type I cell death, or apoptosis, is associated with nuclear and
cytoplasmic condensation and fragmentation, preservation of organelles, and phagocytosis by
neighbouring cells or macrophages. Type II cell death, or autophagic cell death, is characterized
by late nuclear collapse that is preceded by self-degradation of cytoplasmic components by
acidic autophagic vacuoles. While both types of cell death have been associated with normal
development and with human disease, apoptotic cell death has received much more attention
during the past decade. As a result, many molecules associated with apoptosis pathways have
been discovered and studied extensively, and are now being investigated as potential therapeutic
targets. Autophagic cell death, however, remains largely uncharacterized and its associated
molecules and mechanisms in multicellular organisms remain to be elucidated.
        During metamorphosis, the Drosophila larval salivary glands undergo autophagic cell
death that is regulated by a transcriptional hierarchy induced by the steroid hormone 20-
hydroxyecdysone (ecdysone). Known ecdysone-induced and cell death genes have been
implicated in this process through analyses of mutant salivary glands, protein expression, and
gene expression (REFs and Figure 1). Several features of Drosophila salivary gland destruction
make it an ideal system for analyses of gene expression associated with autophagic cell death.
Following a pulse of ecdysone at the prepupal-pupal stage transition, the transcription of several
known and highly conserved cell death genes is upregulated and the entire larval salivary gland
undergoes cell death in a rapid, stage-specific, and virtually synchronous manner. To monitor
autophagic cell death gene expression on a genome-wide scale, we conducted EST and SAGE
analyses of salivary glands dissected from three developmental stages leading up to cell death.
We report here the first comprehensive description of gene expression associated with
autophagic cell death during normal metazoan development.


Results

Tissue-specific Genome-wide Expression

(?Or start with ESTs; we found novel genes, so went to genome-wide analyses)

         To study autophagic cell death in the context of normal development in a multicellular
organism, we examined wild-type patterns of gene expression in Drosophila larval salivary
glands using Serial Analysis of Gene Expression (SAGE). To verify developmental stages for
corresponding SAGE library construction, we dissected salivary glands from several time-points
(every hour from 16 – 24 hr APF at 18C which is equivalent to about 8 – 12 hr APF at 25C) and
analyzed them by staining with the death indicator acridine orange and by quantitative RT-PCR
analyses of the known cell death genes, reaper, hid, and diap2. Based on our results (data not
shown), and those of other studies (Refs; add note about the exception of gene detection at early
stages; sensitivity of Northern vs RT-PCR), we chose three timepoints, 16, 20 and 23hr APF
(18C), for SAGE library construction and analysis. These three time-points displayed
differential expression of known cell death genes and substantial exclusion of acridine orange,
the latter indicative of still-intact cellular membranes (confirm this; ao = ___ agent). SAGE
libraries were constructed, sequenced, and processed to remove low quality sequence, duplicate
ditags, and singletons (see Materials and Methods). Over 30,000 remaining SAGE tags from
each of the three library timepoints were included in further analyses (ref). These tags represent a
total of 4,628 different tags (“tag species”), with ___, ___, and ____ different tag species
present in the 16, 20 and 23 hr libraries, respectively. In each library, the majority of tag species
(>50%) were sampled between 2 – 10 times, while relatively few tag species (__-__%) were
sampled greater than 100 times. This latter group of abundant tags, however, accounts for about
one-half (53%, 53%, and 47% from 16hr, 20 hr, and 23 hr, respectively) of the total number of
tags present in each library, and is not surprising given both the tissue and stage-specific nature
of the starting material.


Tissue-specific ESTs
         To discover new genes and to facilitate specifically the mapping of our salivary gland
SAGE tags to genes, we constructed a cDNA library from 500 pairs of mixed stage (16 hr – 24hr
APF, 18C) salivary glands and conducted 3’ EST sequencing. SAGE tags correspond to the 3’ –
most NlaIII site of their associated transcripts and are often found in 3’ UTR sequence. 5181
high quality salivary gland 3’ EST sequences were clustered and found to represent 1038 + 205
+ 149 different transcripts and 1013 + 205 + 149 different genes (ref). 983 of these transcripts
(or 958 of the genes) matched both BDGP predicted genes and BDGP ESTs and/or cDNAs, 149
matched BDGP ESTs/cDNAs only, 55 matched BDGP predicted genes only, and 205 matched
no gene predictions and no BDGP ESTs/cDNAs. In the latter class, sixteen of the genes
corresponded to EST clusters ( 2) and 189 were present as singletons. These 205 [+ 149 BDGP
est matches] salivary gland genes, or 15 % [ + 149/___] of all genes represented by the salivary
gland ESTs, may be novel genes but could also represent novel splice variants or long 3’ ends of
existing genes. To assess these possibilities further, we determined for each EST the distance to
the nearest gene on the same strand. __% of the ESTs were ___ and __% were far; this latter
group at least likely represent novel genes. All of the salivary gland ESTs except five matched
Drosophila genomic DNA sequence, indicating the comprehensive nature of the Drosophila
genomic DNA resource.
         (Check numbers with erin; split genes, etc.; need to be updated; Not present on an Affy
chip : 205 + 149)

Tag-to-gene mapping in Drosophila
        To identify genes corresponding to SAGE tags, we utilized a Drosophila tag-to-gene
mapping software program built upon an ACEDB style platform (E. Garland et al., submitted/in
preparation?; available at www.--------). To maximize efficiency and accuracy of tag-to-gene
mapping, the program incorporates the use of high quality genomic DNA sequence, full length
cDNA sequences and ESTdata from the Drosophila Genome Project, with the estimation, in the
absence of expression data, of 3’ UTR sequences. In addition, our salivary gland specific 3’
ESTs were added to the database. Tag-to-gene mapping was conducted for each of the three
salivary gland SAGE libraries and we were able to map __% of tag species to known or
predicted genes. __ % of these matches were ambiguous, with tag species assigned to more than
one gene. __ of these SAGE tag species confirmed the existence of predicted genes for which
there is available no other expression data. Of the remaining tag species, an additional __% were
mapped to genomic DNA and/or ESTs not yet associated with a predicted gene (ref). In these
cases, the tag may represent a novel gene or a novel splice form of an already predicted gene, or
the tag may represent a predicted gene whose 3’ end was not sufficiently extended. __ of the tag
species were mapped specifically to our 205 [+149] potentially novel salivary gland ESTs,
demonstrating the complementarity of our tissue-specific 3’ EST/SAGE approach. No sequence
match was found for __ % of tags. Unmapped tags could be due to sequence polymorphisms or
to gaps in the available sequence. A complete list of salivary gland SAGE tag species,
abundance, and mappings can be found at www.supplement.___.


SAGE analysis identifies ecdysone-induced and cell death genes associated previously with
salivary gland PCD
        Several ecdysone-induced genes and cell death genes associated with Drosophila larval
salivary gland death have been described previously (Figure 1 pathway and refs). As anticipated,
we found tags corresponding to several of these genes in our SAGE libraries and Figure 1
indicates their relative expression levels (indicate significance in the figure legend). BR-C, E74
and E75 are general ecdysone-induced primary response genes shown previously to regulate
salivary gland expression of both rpr and hid, hid only, and diap2, respectively (Jiang et al. 2000;
see Baehrecke reference). E93 is a stage and tissue-specific ecdysone-induced primary response
gene required for maximal expression in prepupal salivary glands of BR-C, E74A, E75A and the
cell death genes rpr, hid, ark, and crq (Lee et al. 2000). Consistent with previous reports, we
detected increased expression from 16 to 23 hrs APF of the pro-death genes Ark, dronc and crq.
Genes expressed at insignificant levels, E93, reaper and diap2, were analyzed further by
quantitative RT-PCR. By this method, E93 expression peaked at __ hrs APF, reaper expression
peaked at 23 hrs APF, and expression of diap2, a cell death inhibitor, decreased slightly from 16
to 23 hrs APF. Genes known to be expressed during salivary gland PCD but not detected in our
libraries are Ecr, USP, BFTZ-F1 and hid. These genes all possess putative NlaIII recognition
sites and thus theoretically can be associated with a SAGE tag. However, Ecr, USP and BFTZ-
F1 act upstream of the primary response genes described above and thus may be expressed
maximally prior to 16 hrs APF. This interpretation is consistent with Northern analysis of
BFTZ-F1 (Jiang et al. 2000). Alternatively, these genes may be expressed at very low levels.
Failure to detect hid was not surprising because other detection methods indicate it is expressed
at levels lower than reaper (Ref, data not shown). In general, the gene expression profiles
generated by SAGE are consistent with previous reports and can temporally distinguish known
upstream transcriptional regulators from downstream death effector molecules.
[or are we looking too early; test by rt-pcr; second ecdysone pulse at about 10 hr APF]


Many genes not previously associated with salivary gland PCD are differentially expressed
        We conducted pairwise comparisons between the SAGE libraries (ref) to identify
additional genes expressed differentially prior to salivary gland PCD. In the 16 hour versus 23
hour comparison, we found 522 genes significantly upregulated and 331 genes significantly
downregulated. Together, these genes account for 20% of all genes expressed in the salivary
glands during these two stages. In the 16 hr versus 20 hr and 20 hr versus 23 hour comparisons,
we found xxx and xxx genes significantly upregulated as well as xxx and xxx genes
significantly downregulated, respectively. A complete list of differentially expressed genes is
available at Supplementary data (www.-------). Table 2 includes a subset of differentially
expressed genes annotated previously with respect to function (REF GO), sorted by functional
category. While we have included genes from multiple comparisons, emphasis is placed on
genes upregulated between 16 and 23hrs APF, the profile describing the majority of known cell
death genes. In the instances where a differentially expressed SAGE tag matched a potentially
novel gene (i.e. not predicted), we used BLAST analysis to determine any possible sequence
similarities to genes of known function (REF). Biological and molecular function annotations
are Gene Ontology terms from FlyBase, except where indicated otherwise. Since some known
cell death related genes, e.g. reaper, were detected at low levels in our libraries, we also
conducted direct searches of all SAGE tags and ESTs for genes predicted to play a role in cell
death (REF Aravind, Flybase keyword, other). Below is a summary of our findings:
        Make a chromosome map of the upregulated and downregulated genes?

Ecdysone induced and hormone-related genes
In addition to BR-C, E74, E75 and E93, we identified additional ecdysone-induced genes that
were differentially expressed in the salivary gland libraries (graph or Table). Highly abundant
were members of the L71, or Eig71E, late gene family. The function of the L71 genes is not
well known? but they are reported to be induced in late third instar larvae. Their abundance at
16 hr APF and decline by 23 hr APF is consistent with a role during the early larval ecdysone
pulse ( ). Eip63E (serine-threonine protein kinase) was detected at 16 hr APF at levels similar to
BR-C. What is known about 63E? , . Eip63F-1 (calcium binding EF-hand family member) and
Eip71CD (Eip28, protein-methionine-S-oxide reductase) both peaked in expression at 20 hr
APF, indicating a possible role for these genes in salivary gland cell death regulation. See
review; not described here before; possible new role??

Include Hr78 – negative regulator?



Cell death genes

Autophagy associated genes : known indicators (galactosidase, e.g.)

Cytoskeleton associated genes
actin, vimentin, microtubule-related.
See cytoskeletal paper from german group; name of gene? Discs large?

Proteases: Cathepsins, caspases, serine proteases

Lysosomal:

Proteasome:

Ubiquitin:

Signal Transduction

Metabolism

Ribosomal (see reference - )

Unknowns
Data mining by automated text and sequence similarity based queries

Data mining identifies additional putative Drosophila orthologs of mammalian cell death-
related and disease-associated proteins


        Genome-wide analyses produce large amounts of data that cannot be effectively analyzed
manually. To annotate further the set of genes expressed prior to Drosophila salivary gland
death, we developed automated data mining tools that incorporate publicly available databases
(Figure x). Salivary gland ESTs or SAGE tags with a corresponding gene product in the
GadFly___ database were analyzed further by sequence similarity and user-defined keyword
queries of the Swissprot (or Trembl; see ref) database. This analysis allowed us to identify
differentially expressed genes with protein products similar to those from any other organism
and also associated with a specific property, e.g. apoptosis, of interest. An example of our data
mining results is shown in Table __ and a complete list is available at www. _____. This
analysis allowed us to find, in our large data set, genes associated previously with cell death,
apoptosis, and cancer, for example, and also led to the discovery of novel associations with such
processes. For example, __________________

         To investigate further the salivary gland __ ESTs and __ differentially expressed SAGE
tags (with unambiguous matches; or include all novel ESTs here) corresponding to novel genes,
we conducted tBLASTX analyses and PFAM domain searches of matching ESTs and
surrounding genomic DNA sequence [Ref.; N.B. want to consider only same strand hits. For the
latter, we extracted sequence 2500 bp upstream of our EST or SAGE tag, and 500 bp
downstream of the SAGE tag. Only matches corresponding to sequence on the same strand as
the SAGE tag were considered.] For __ of the genes, we detected matches to proteins with e <
____ (Table __ and www.___ ). All matching proteins were further analyzed by keyword
queries. Of particular note was ____________________.




Verification of SAGE data by duplicate libraries and real-time quantitative RT-PCR
         To assess the reproducibility of SAGE library construction, we prepared independently
two SAGE libraries from the 16 hr APF timepoint. All steps of SAGE library preparation,
including tissue dissection, were conducted separately. Following sequencing and tag processing
(ref), a comparison of the libraries indicated insignificant differences in expression for __ % of
the tags. The observed differences (__%), therefore, are consistent with the number expected to
occur by chance. To verify differential expression between libraries determined by SAGE, we
conducted real-time RT-PCR analyses for __ differentially expressed genes representing the
complete range of p values (abundance? Fold-difference?). Up or downward trend vs. fold-
difference. The fold difference in expression achieved by SAGE and real-time RT-PCR was
compared and is indicated graphically for the 16 and 23 hr timepoints in Figure __. (complete
data set available at www.___). Overall, we confirmed differential expression for __ of the ___
genes tested. Did we detect a diff btn genes expressed at high levels and those expressed at
lower levels?


Mutant Analysis identifies E93 regulated genes in salivary gland cell death
        The ecdysone-induced genes, ____ _______ are considered general regulators of
ecdysone-induced processes but E93 expression appears to specifically foreshadow steroid-
induced cell death. E93 encodes a novel nuclear protein that binds to multiple specific sites on
larval salivary gland polytene chromosomes. In E93 mutants, salivary glands persist and the
expression of E74, __?, Ark, dronc, rpr, hid, and crq fails to be upregulated normally. The map
positions of __ and crq correspond to E93 binding sites and may thus be regulated directly by
E93. To identify other genes that may be regulated transcriptionally by E93 in salivary gland
death, we screened all differentially expressed genes for those with a map position corresponding
to E93 binding sites. We identified ___ upregulated genes and ___ downregulated genes
corresponding to ___ of the ___ known E93 binding sites (Table?). to test further whether they
may be regulated directly by E93, we tested a subset to see whether they fail to be upregulated.
Since previous evidence indicates a role for E93 as a positive regulator, we tested genes
upregulated at 23 hrs apf. A subset of the genes upregulated at 23 hrs APF was



Secondary screen for general regulators and effectors of programmed cell death
        The expression of the known pro-death genes reaper, hid, crq, and dronc (?),.forshadows
death not only in the salivary gland but in other tissues as well. To identify genes with a possible
role in cell death beyond the salivary gland, we examined the expression of upregulated salivary
gland genes in cell death stage embryos. During embryonic development, apoptotic cell death
occurs in reproducible spatial and temporal patterns. Cell death is predominant in the head
region and cephalic folds during stage 12, and in the ventral nervous system and midline (?)
during stages 15 and 16 (Ref; confirm).




 Autophagy not described (describe as single cells versus whole tissues). during stage 12 and
_____ during other tissues that die. The expression of the known death genes,,,,, forshadows
death of not only the salivary gland but of other tissues as well. In the embryo, for example,
specific pattern of cells die…; described morphologically as apoptotic? … We initiated a pilot
screen of __ genes if we could identify additional genes in our set that might not only salivary
gland death but also that of dying cells Known cell death regulators such as reaper, hid, dronc?
And sickle are transcriptioally upregulated prior to salivary gland death and also prior
tupregulated prior to salivary gland death differentially upregulated genes ene expression in other
tissues that die. The expression of known cell death genes; positive death regulators/effectors ,
reaper,hid, sickle, dronc? Precedes the death of cells in the embryo and produces a specific
reproducible pattern of expression….To screen for additional genes like reaper, hid, ark, crq,
dredd, and sickle? that have a general role in cell death, as opposed to a specific role in
autophagic salivary gland death, we investigated gene expression in other tissues that die. Cell
death in the embryo …. Describe. thathelp identify genes with a general role in cell death
Several known cell death genes ; we described sickle, dredd, others? Given the overlap of known
death genes in both autophagic and apoptotic cell death …..We initiated a pilot screen aimed at
identifying additional genes that are transcriptionally regulated in both autophagic and apoptotic
cell death. Are genes transcriptionally upregulated in salivary gland death similarly associated
with other tissues undergoing cell death? Cell death in embryos investigate whether genes
transcriptionally regulated prior to autophagic cell death in the salivary gland are also
upregulated prior to apoptotic cell death in other Drosophila tissues, as is the case for reaper, hid,
ark, and dronc, we

Summarize this section with a statement about tissue specific (autophagy?) vs general/ apoptotic


Autophagy and Human Disease

       Autophagy (autophagic cell death?) has been associated previously with
neurodegenerative disease, cancer, and _____. To investigate these and other possible
associations between autophagic cell death and human disease, we searched differentially
expressed genes for OMIM similarities….the Homophila Database for genes differentially
expressed in the salivary gland libraries. Homophila combines…..describe

Other:

In addition to these genes, we found salivary gland expression of the previously characterized
death genes diap1, dredd, and sickle. (also Buffy?). Other studies using Northern blot analyses
of salivary gland RNA indicated that diap1 and dredd were not expressed in salivary glands at
these stages. We confirmed their expression by Q RT-PCR. Our analyses indicate the
sensitivity of the SAGE method (but why is dredd higher than rpr then? Need to confirm dredd
and diap1). Say something about previous association with apoptosis; also autophagic cell
death; overlap.
We did not find hid; low levels relative to rpr; stability of message?

In addition to genes associated previously with salivary gland death, we identified known
ecdysone-induced and known cell death genes not yet associated with cell death in this tissue.
(and ecdysone-regulated genes?)
Caspases involved? E93 ref; dredd not expressed
Mention the abundant L71 genes here also.
We searched for genes with cell death related domains as predicted by Aravind et al. (reference
the database). Our data provides supportive evidence for a role in autophagic cell death (see
below; in situs also). Mention Aravind genes in the uncharacterized section; ie. Predictedonly;
e.g. BNIP
        __ of the known ecdysone-regulated and __ of the cell death genes were differentially
expressed (p < 0.05; Ref). Define and Summarize the differential expression. Some genes not
detected at levels sufficiently high to determine statistically whether genes are differentially
expressed. Deeper sequencing to conclude statistically. Test by real-time RT-PCR.
% matched to known/predicted genes; how many gene predictions do we confirm? Ie. CG# with
no associated ESTs?
(these would be in our genes category; can we determine how many have no associated
ESTs/transcript info?)
% matched to ESTs (BDGP)
% matched to ESG01 ESTs only
% No tag match found
-percent of genes with ambiguous tag match ( calculated vs. observed)
        -make a statement about the completeness of the DGC data set.

Figures/Tables:

   1. AO and RT-PCR of 18C salivary glands (or describe)
   2. SAGE tag summary table (or describe)
   3. Tag-to-gene mapping summary table (or describe)
   4. Known cell death genes; table or bar graph
   5. Differentially expressed genes; summary table; large table via web
   6. Verification data (QRT-PCR); Table or graph
   7. Embryo in situs/TUNEL
   8. RT-PCR in mutants (could combine with verification RT-PCR table)
   9. Swissprot/TrEMBL keyword search summary table
   10. Database structure diagram


Materials and Methods

Fly strains: The wild-type Drosophila melanogaster strain used was OreR, obtained from the
Bloomington Drosophila stock center. To reduce any possible sequence variability, a single pair
mating from the OreR stock was used to construct an OreR[SAGE] line. For mutant analyses,
non-Tubby pupae were selected from progeny of a cross between E93[1]/TM6B and w;
Df(3R)93F[x2]/TM6B,Hu,Tb,e. Stocks were maintained at room temperature and then shifted
to 18C following collection of white prepuae (time 0, hours after puparium formation).

Tissue dissection and RNA preparation: Salivary glands were dissected from OreR[SAGE] and
E93 mutant animals at various stages of development (hours after puparium formation at 18C).
Timepoint determination here?equivalent to 25C timepoints. Salivary glands were dissected into
PBS and then immediately transferred to RNAlater (Ambion __). Tissue was stored in RNAlater
at 4C overnight and then transferred to –20C. Total RNA was extracted using Trizol and treated
with DNAse (____).

CDNA library construction and EST sequencing:
SAGE library construction: SAGE libraries were constructed with the Invitrogen I-SAGE kit
using __ ug of OreR[SAGE] salivary gland total RNA as the starting material. Four SAGE
libraries were prepared from salivary glands dissected at three different timepoints: two libraries
from 16 hr APF (sg16a and sg16b) , one from 20 hr APF (sg20) and one from 23 hr APF (sg23).
Modifications to the manufacturer’s protocol and supplies included the use of biotinylated
linkers (detail), ___ other?. Ditags were obtained by amplification for __ cycles, __ cycles,
__cycles, and __ cycles, respectively. PCR rounds, etc. Report # of PCR cycles, vector,
bacterial strain used for transformation.

SAGE sequencing and data processing: DNA sequencing was conducted on an ABI 3700
sequencer using __ primer (seq) and ABI BigDye termination reaction mix (find out exact
protocol; amount of mix used?). Sequence was retrieved, quality assessment, vector removal
using ____. SAGE ditags were removed using ___ duplicate ditags were removed. Unique
ditags were processed to recover individual 10 bp tags (+ CATG for 14 bp total). Removal of
tags with less than 95% overall sequence quality.

(**Ref of protocol – Steve’s paper? Cryptococcus paper?)

SAGE data analyses: SAGE tags from the three libraries were compared and all tags present less
than two times in all three libraries combined (i.e. singletons) were excluded from further
analyses. SAGE libraries were compared pairwise using an in-house database called SAGEdb
and p values were calculated using the method of Audic and Claverie (REF). Statistical
analyses used.

Database mining:

Tag-to-gene mapping:
Probe preparation and embryo in situ hybridization: Single stranded digoxigenin-labeled DNA
probes were prepared essentially as described (? REF) except that the template was generated by
reverse transcription (Kit name, company) from embryo or salivary gland RNA using
oligonucleotide primers synthesized from ______. Probe templates were verified by end
sequencing (to do). Both antisense and sense control DNA probes were prepared for each gene
tested. A list of primer sequences used can be obtained from www._______. In situ
hybridization was carried out as described in ______ with Drosophila embryos collected
essentially as described in ____.
Salivary gland in situs?

Real-time RT-PCR: Real-time RT-PCR was performed on an Applied Biosystems 7900 _____.
ABI Sybr Green One-step RT-PCR------ reagent was used with 50 ng RNA template, and ___
primer. A list of primer sequences used is available at www.-------. Drosophila rp49 was used as
a control for RNA levels and for sample normalization following the method outlined in ______.


For Abstract/Intro:

Lysosomal componenets, pathways,….
First comprehensive molecular description of metazoan autophagic cell death
Ded, rip and bh3-only
Data mining identifies similarities to XX cancer-related genes, providing
Autophagy implicated in what diseases
Overlaps with apoptotic cell death are substantial; provide potential new markers for autophagic
cell death; example of an unknown gene; human homolog; differentially expressed as in beclin-
1???
Core Apoptotic genes also associated with autophagic cell death pathways; implications for
therapeutic target consideration
Members of TNF pathway not described before in Dm
** BUT death in embryo = autophagy??
Novel ESTs; correspond to diff expressed SAGE tags (how many – sequence the 5’ ends of
these?; any with interesting similarities to disease, death related genes??
Together, the ESTs and SAGE tags confirm xx predicted genes and identify xx novel genes; xx
of these are differentially expressed prior to salivary gland PCD

				
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