Survey of transcripts in the adult Drosophila brain.

BACKGROUND: Classic methods of identifying genes involved in neural function include the laborious process of behavioral screening of mutagenized flies and then rescreening candidate lines for pleiotropic effects due to developmental defects. To accelerate the molecular analysis of brain function in Drosophila we constructed a cDNA library exclusively from adult brains. Our goal was to begin to develop a catalog of transcripts expressed in the brain. These transcripts are expected to contain a higher proportion of clones that are involved in neuronal function.
RESULTS: The library contains approximately 6.75 million independent clones. From our initial characterization of 271 randomly chosen clones, we expect that approximately 11% of the clones in this library will identify transcribed sequences not found in expressed sequence tag databases. Furthermore, 15% of these 271 clones are not among the 13,601 predicted Drosophila genes.
CONCLUSIONS: Our analysis of this unique Drosophila brain library suggests that the number of genes may be underestimated in this organism. This work complements the Drosophila genome project by providing information that facilitates more complete annotation of the genomic sequence. This library should be a useful resource that will help in determining how basic brain functions operate at the molecular level.

Background

Drosophila melanogaster is an important model organism. After more than 50 years of study, the anatomy of the brain is well described and many brain functions have been mapped to particular substructures [1,2,3,4,5,6,7,8]. The adult brain is composed of approximately 200,000 neurons which are organized into discrete substructures. The optic lobe (composed of the lamina, medulla, lobula and lobula plate) is primarily involved in the processing of visual information from the photoreceptors and sending that information to the central brain [2,5,9]. The antennal lobes are chiefly responsible for the processing of olfactory information [10]. The mushroom bodies are involved in olfactory learning and memory and other complex behaviors [11,12,13,14,15]. A group of approximately six neurons in the lateral protocerebrum are sufficient to drive circadian rhythms in locomoter activity [16,17]. The central complex, although poorly understood, appears to be involved in motor coordination [18,19,20].

Despite our increasing knowledge of Drosophila brain anatomy and function, relatively little information is available concerning the molecules expressed in the brain that coordinate function and manifest behavior. Classic methods of identifying genes involved in neural function include behavioral screening of mutagenized flies, then rescreening candidate lines for pleiotropic effects due to developmental defects. This process is both laborious and time consuming. To augment this genetic approach, sequencing of random cDNAs is proving effective in identifying genes expressed in a specific cell type [21]. Much information has been collected through the analysis of expressed sequence tags (ESTs) [22,23,24,25]. Using this approach, sequence information is gathered from one or both ends of a cDNA and cataloged to determine the complexity of an mRNA population. Here, we use a modified EST approach and completely sequence novel cDNAs. Others have used a similar approach by shotgun sequencing concatenated cDNA inserts [26,27]. One goal of our work was to begin to develop a catalog of transcripts expressed in the brain. These transcripts, because of the location of their expression, are expected to contain a higher proportion of clones that are involved in neuronal function.

Many Drosophila head libraries have been used to isolate cDNAs that correspond to genes identified by genetic screens for their involvement in brain function. Several transcripts identified in this manner are expressed at a relatively low level (dunce [28], CREB [29], dco [30], period [31], timeless [32], dissonance [33]). The Drosophila brain makes up only a small part of head tissue (approximately 14% dry weight). By eliminating non-brain tissues, we increase the relative representation of rare neural transcripts in this unique library.

We began a catalog of the genes expressed in the brain of adult Drosophila in support of more conventional methods of understanding brain function. Cataloging sequence information and publishing the data through electronic databases has enriched molecular science in general. In a matter of a few minutes, one can use information from a single sequencing reaction to identify a gene that was sequenced by another laboratory, and one maybe able to deduce the function of the isolated clone. This set of tools facilitates molecular work in virtually every branch of biological sciences. This report details construction, quality analysis and initial characterization of a unique library created from adult Drosophila brains. Surprisingly, we discovered that 11% (29 clones) of the Drosophila brain cDNA clones that were randomly chosen for analysis are not matched with any EST sequence generated in support of the Drosophila genome project (as of 10 October 2000). Further, the genes encoding 59% of these novel ESTs are not predicted by algorithms used for fly genome annotation. From our analysis of ESTs that do not correspond to one of the 13,601 annotated genes, we predict that the number of genes in the Drosophila genome may be underestimated by 10-15% (approximately 1,300 to 2,000 genes).

Results and discussion

Library quality assessment

Desiccated brain tissue from adult Drosophila melanogaster was used to construct a library using the Stratagene Hybrid-Zap system. This library was designed for protein expression and, therefore, was constructed such that full-length cDNAs containing 5' untranslated regions are not likely to be present. The number of clones in the library and the size of the clones were used to assess the quality of the library. The number of clones in the primary library was determined by titering one of the five packaging reactions. The total number of clones in the primary library is 6.75 × 106 (that is, all five packaging reactions). From the analysis of the fully sequenced clones (141 novel and matched isolog clones reported in this study), the majority of the inserts (53%) were between 400 and 800 base pairs (511 base pairs ± 197 base pairs average deviation). Characterized clones from the library range between 139 to 1,746 base pairs (bp), including only 15 As of the poly(A) tail. The insert size for this library is as expected using the Stratagene Hybrid-Zap kit, given that the size-selection column retains DNA molecules larger than 200 bp) (Stratagene technical support, personal communication). Of the 283 clones that were either completely or end-tagged sequenced, approximately 4% (12 clones) did not contain an insert (Figure 1).

Figure 1

Scheme for classifying the Drosophila brain library clones.

Clone selection

To try to maximize the discovery of novel transcripts, we investigated whether there was a correlation between transcript abundance and the presence of the sequence in a public database. Specifically, a reverse northern blot experiment using radiolabeled head cDNA was performed to determine whether hybridization level could be used to identify frequently occurring transcripts. We reasoned that the abundance of these transcripts may increase their representation in data banks when compared to less abundant transcripts. The data from this experiment are shown in Table 1.

Table 1

Hybridization data from 85 randomly chosen clones

Hybridization level	Originally novel	Signal
Absent	AF171761	242.2
	AF171771	282.1
	AF171773	212.4
	AF171774	264.8
	AF171776	262.6
	AF171777	293.6
	AF171778	296.6
	AF171781	299.4
	AF171762	194.0
	AF179229	211.9
Light	AF171764	428.0
	AF171765	461.4
	AF171769	483.8
	AF171772	305.0
	AF171779	460.6
	AF171782	335.6
	AF171785	360.3
	AF179230	444.6
Medium	AF171763	508.5
	AF171766	617.2
	AF171767	615.6
	AF171768	541.3
	AF171770	509.0
	AF171786	681.5
	AF171787	924.1
	AF171789	984.0
	AF171790	978.2
	AF171791	862.7
	AF171792	583.2
	AF171793	731.2
	AF171794	695.5
Dark	AF171762	1237.0
	AF171775	1066.0
Hybridization level	Known	Signal
Absent	None
Light	AF171706 Drosophila GS2 for glutamine synthase	315.3
	AF171707 Drosophila ubiquitin protein gene	388.6
	AF171709 Drosophila cytochrome c oxidase	365.3
	AF171711 Drosophila calmodulin gene	340.7
	AF171715 Drosophila CCATT box-transmembrane domain	392.1
Medium	AF083504 Drosophila Pls dso3465 (d149) dso 8544 (D187)	645.3
	AF171701 Drosophila frequenin gene	503.4
	AF171702 Drosophila nicotinic acetylcholine receptor	526.0
	AF171704 Drosophila ADP/ATP Translocase	537.1
	AF171705 Drosophila tyrosine kinase gene	544.5
	AF171703 Drosophila ferritin subunit 1 (Fer1) mRNA	552.0
	AF171708 Drosophila mRNA for rab-related protein 4	543.2
	AF171710 Drosophila cytochrome c oxidase subunit	538.0
	AF083505 Drosophila 2-g8 from P1 DS02782 (D71)	870.4
	AF171712 Drosophila gene encoding S-adenosylmethionine decarboxylase	918.0
	AF171713 Drosophila TRIP-1 homolog (Dm TRIP) mRNA	576.6
	AF171714 Drosophila twinstar (tsr) gene	664.1
	AF171716 Drosophila burdock retrotransposon gag protein	631.6
	AF171717 Drosophila GS1 mRNA for glutamine synthase	557.8
	AF171718 Drosophila geranylgeranyl transferase	694.9
Dark	None
Hybridization level	Ribosomal/mitochondrial	Signal
Absent	AF083279 Ribosomal protein rat 60s L35A	235.1
	AF083516 Drosophila ribosomal protein S17 gene	299.2
	AF083518 Drosophila ribosomal protein L31	218.4
Light	AF083272 Yeast ribosomal protein L46	489.6
	Clone 17 Mitochondrial 16S ribosomal mRNA	480.4
	Clone 26 Mitochondrial 16S ribosomal mRNA	343.2
	AF083276 M. musculus ribosomal protein L21 mRNA	361.4
	AF083277 M. musculus ribosomal protein L21 mRNA	388.1
	AF083278 Ribosomal protein human 60s L24	326.8
	AF083515 Drosophila ribosomal protein 15a (40s subunit)	376.5
	Clone 61 Mitochondrial 16S ribosomal mRNA	354.1
	Clone 72 Mitochondrial 16S ribosomal mRNA	470.5
	AF083520 Drosophila ribosomal protein L18a	352.6
	AF083521 Drosophila ribosomal protein S14 A and B genes	396.5
Medium	AF083513 Drosophila mRNA ribosomal protein	508.8
	AF083514 Drosophila ribosomal protein L19 gene	579.8
	AF083281 Ribosomal protein R. norvegicus S23	798.1
	AF083519 Drosophila 60S ribosomal protein L43 mRNA	837.9
	Clone 80 Mitochondrial 16S ribosomal mRNA	601.1
	Clone 90 Mitochondrial 16S ribosomal mRNA	798.6
	AF083522 Drosophila 5.8S and 2S ribosomal rRNA	819.8
	Clone 94 Mitochondrial 16S ribosomal mRNA	820.0
Dark	AF083275 Drosophila ribosomal protein S18 mRNA	1166.0
	AF083517 Drosophila ribosomal protein L22 mRNA	2423.0
Hybridization level	Isologs	Known
Absent	AF083295 C. pothophila cytochrome oxidase I & II	224.9
	AF083300 Human clathrin coat-associated protein 50	233.7
Light	AF083301 R. norvegicus trg mRNA carrier protein precursor	474.5
Medium	AF083296 Human protein synthesis factor (eIF-1A)	512.2
	AF083298 Silkworm mRNA for DNA SC factor	500.1
	AF083297 Bovine ATP synthase G chain, mitochondrial, H+ transporting	555.9
	AF083299 H. sapiens Arp 2/3 complex 20 kD subunit, actin related protein	570.4
	AF083302 H. sapiens mRNA for testican	519.7
Dark	None

The photo stimulating units (psl) within a circle of standard area was used to determine the relative hybridization level to radiolabeled head cDNA for each clone. Hybridization categories are as follows. Absent, 0-300 psl (corresponding to background); light, 301-499 psl; medium, 500-899 psl; dark, 900-2500 psl. Clones are categorized as follows. Novel, sequence information for clones that were novel at the initial phase of this project; known, Drosophila sequence information previously submitted to sequence databanks; ribosomal protein/ribosomal RNA/mitochondrial, sequences corresponding to ribosomal proteins, ribosomal RNAs or mitochondrial transcripts; isologs, transcripts that have a high degree of similarity to sequences reported in the databanks.

The level of hybridization to the probe varied considerably within a category. In particular, novel transcripts did not uniformly have low levels of hybridization, which suggested that hybridization level would not greatly aid in identifying novel clones. Therefore, subsequent clones for this study were randomly chosen for sequence analysis. It is possible that abundant transcripts may not be as well represented in the database as a result of directed cloning of rarer molecules, or that cDNA abundance in this library may not accurately reflect relative transcript abundance in the fly brain.

Sequence data

We obtained sequence data for 271 independently isolated cDNAs representing transcripts expressed in the Drosophila brain (Figure 1, Table 2). Of these, 141 clones originally classified as either novel (114 clones) or matched isologs (27 clones) were completely sequenced. Only end-tag sequence data was collected for clones classified as matched isolog ribosomal protein sequences (16 clones), known Drosophila sequences (71 clones) and known Drosophila ribosomal protein sequences (23 clones). All insert sequences or ESTs can be obtained by searching GenBank with the appropriate accession numbers listed in Table 2. Data for 20 mitochondrial 16S clones are not reported here because mitochondrial expression is not the focus of this study.

Table 2

GenBank accession numbers of all sequenced clones

Clone category	GenBank accession number	Total
Novel sequences only matched to genomic data	AF171764, AF171789, AF171794, AF171800, AF171805, AF171808, AF171813, AF171815, AF171819, AF171821, AF171828, AF171838, AF171850, AF171854, AF171858, AF171859, AF171865.	17
Matched with an EST, but NOT a predicted gene	AF171766, AF171772, AF171779, AF171780, AF171781, AF171782, AF171785, AF171787, AF171796, AF171797, AF171804, AF171811, AF171818, AF171826, AF171829, AF171837, AF171839, AF171840, AF171845, AF171848, AF171857, AF171860, AF171862, AF171863, AF171869.	25
Matched with a predicted gene, but NOT an EST	AF171867, AF171768, AF171778, AF171762, AF171790, AF171799, AF171771, AF171803, AF171812, AF171832, AF171861, AF171868.	12
Matched with an EST and a predicted gene	AF171761, AF171762, AF171763, AF171765, AF171767, AF171769, AF171770, AF171773, AF171774, AF171775, AF171776, AF171777, AF171784, AF171786, AF171788, AF171791, AF171792, AF171793, AF171795, AF171798, AF171801, AF171802, AF171806, AF171807, AF171809, AF171810, AF171814, AF171816, AF171817, AF171820, AF171822, AF171823, AF171824, AF171825, AF171827, AF171830, AF171831, AF171833, AF171834, AF171835, AF171836, AF171841, AF171842, AF171843, AF171844, AF171846, AF171847, AF171849, AF171851, AF171852, AF171853, AF171855, AF171856, AF171864, AF171866, AF171870, AF171871, AF171872, AF179229, AF179230.	60
Matched isolog	AF083295-AF08321.	27
Matched isolog ribosomal protein sequences	AF083272, AF083275-AF083279, AF083281, AF083286- AF083294.	16
Known Drosophila ribosomal protein sequences	AF083513-AF083522, AF083524-AF083526, AF083528, AF083530, AF083531, AF083537, AF083538, AF083544-AF083548.	23
Known Drosophila sequences	AF083504-AF083512, AF171701-AF171743, AF171745-AF171760, AF171784, AF171807, AF171872.	71

Sequences classified as 'novel' represent new EST sequence data from D.melanogaster (as of 10 October, 2000)and are not homologous to any EST or cDNA sequence in GenBank. These 17 novel sequences are not predicted to be transcribed, and are described in more detail in Table 5b. Sequences classified as 'matched with an EST' correspond to known EST sequence data, but do not have a corresponding predicted gene. Sequences classified as 'matched with a predicted gene' correspond to those that are predicted genes, but do not have corresponding EST data. Sequences classified as 'matched with an EST and a predicted gene' have both EST and predicted gene matches. Sequences classified as 'matched isologs' are sequences that are homologous to genes found in other organisms. Sequences classified as 'matched isolog ribosomal protein' are sequences that are homologous to ribosomal protein genes found in other organisms. Sequences classified as 'known Drosophila ribosomal protein sequences' are matched with sequences previously reported to GenBank. Sequences classified as 'known Drosophila' are sequences that have been previously reported to databases (AF083507 and AF083508 correspond to clone 159; AF083509 and AF083510 correspond to clone 226).

Table 5b

Additional information

Accession number	EST hit?	Predicted gene?	AAATAA/Poly(A) spacing	Splicing detected?	Expression detected in	Insert size	Comments
AF171819	NO	NO	37		Body	145
AF171858	NO	NO	18		Body	186
AF171764	NO	NO	280		Both	450
AF171805	NO	NO	25		Both	259
AF171789	NO	NO	134		Brain	857
AF171794	NO	NO	45		Brain	610
AF171800	NO	NO	Not present		Brain	190
AF171808	NO	NO	23		Brain	269
AF171815	NO	NO	12		Brain	363
AF171854	NO	NO	52	Spliced, consensus	Brain	195	Extensive poly(A) tail (146 nucleotides)
AF171813	NO	NO	Not present		Neither	216	Extensive poly(A) tail (>42 nucleotides)
AF171821	NO	NO	Not present	Spliced, consensus	Neither	359
AF171828	NO	NO	Not present		Neither	189
AF171838	NO	NO	18		Neither	430
AF171850	NO	NO	13		Neither	1104
AF171859	NO	NO	18		Neither	1786
AF171865	NO	NO	Not present		Neither	452

Additional information concerning clones that lack EST data and that are not predicted to be transcribed (Novel cDNA). The GenBank accession number for each of the 17 clones is indicated. The distance between a putative poly(A) addition sequence (AAATAAA), when present, and the poly(A) sequence is shown. 'Splicing detected?' indicates the two cDNA clones showing evidence for consensus splicing. 'Expression detected' refers to each clone's hybridization results when probed with radiolabeled cDNA from brain or body (see Table 6). 'Insert size', size of the cDNA insert; 'comments', additional comments for the identified clone. None of the 17 cDNA is predicted to encode a protein larger than 100 amino acids.

We generated sequence data for 27 Drosophila genes that had been previously sequenced from other organisms. Isologs that were identified in the brain library but which had not been identified or sequenced in Drosophila melanogaster are listed in Table 3 (with the exception of ribosomal protein genes). An isolog is defined as a sequence that has a high degree of similarity to genes identified in other organisms, but the functional relationship between these genes has not been demonstrated [34]. As expected, we recovered many previously identified Drosophila genes (Table 4). We did not continue with full insert sequencing of these Drosophila sequences, but the EST data for these clones was submitted to GenBank.

Table 3

Isologs identified in Drosophila brain study

Gene	Organism	Accession Number	Score	Probability
Cytochrome oxidase I and II	C. pothophila	AF083295	201	6.2e-8
Protein synthesis factor eIF-1A	H. sapiens	AF083296	237	1.1e-23
ATP synthase G chain	B. taurus	AF083297	282	2.6e-30
DNA supercoiling factor	Silkworm	AF083298	249	1e-65
Arp2/3 complex 20 kD subunit	H. sapiens	AF083299	681	3.7e-85
Clathrin coat-associated protein 50	H. sapiens	AF083300	663	1.5e-85
Trg gene	R. norvegicus	AF083301	300	7.1e-39
Testican gene	H. sapiens	AF083302	806	8.8e-57
Calmodulin-like processed pseudogene (similar to D. melanogaster DMTnc 73F troponin but not identical)	H. sapiens	AF083303	123	1.0e-27
Peripheral type benzadiazipine receptor	H. sapiens	AF083304	220	5.3e-38
Retinal protein 4	H. sapiens	AF083305	459	4.2e-73
Ubiquitin-like S30 ribosomal fusion protein	H. sapiens	AF083306	151	1.9e-15
Insulinoma rig-analog DNA-binding protein	H. sapiens	AF083307	498	1.3e-31
Neuronal calcium binding protein	C. elegans	AF083308	629	8.2e-78
Mitochondrial ubiquinone-binding protein	H. sapiens	AF083309	115	2.0e-25
Oxidoreductase gene	H. sapiens	AF083310	172	3.4e-23
Iron-sulfur protein	R. rieske	AF083311	812	5.1e-57
Copper chaperone for superoxide dismutase	H. sapiens	AF083312	359	2.0e-42
Putative fatty-acid binding protein	A. gambiae	AF083313	530	1.0e-53
SMT3 protein	H. sapiens	AF083314	649	8.3e-44
Core P2 precursor ubiquinol cytochrome c reductase complex	B. taurus	AF083315	172	5.5e-15
Tyrosyl-tRNA synthetase	H. sapiens	AF083316	500	7.8e-39
Transferrin gene	Flesh fly	AF083317	511	1.0e-61
Leucyl-tRNA synthetase	A. thaliana	AF083318	179	3.1e-72
Protein translation factor SUI1	A. gambiae	AF083319	381	1.8e-47
Uracil phosphoribosyl transferase	S. cerevisiae	AF083320	434	8.7e-51
Metallopanstimulin gene	H. sapiens	AF083321	322	3.3e-39

'Gene' indicates the homologous gene name, 'organism' indicates the organism which has the greatest similarity to the Drosophila clone, and the accession number for the Drosophila isolog is listed. The 'score' and 'probability' for each match using BLASTN [41] are reported.

Table 4

Brain cDNA clones matched with previously reported Drosophila genes

Gene	Accession number	Location	Reference
Frequenin gene	AF171701	CNS and PNS of adults and embryos	[45]
Ferritin subunit 1 gene	AF171703	Fat body and gut of larvae, present in all stages and increased with iron supplementation	[46]
Tyrosine kinase gene	AF171705	Not specified	-
Glutamine synthase gene	AF171706	Mitochondrial
	AF171717
Rab-related protein 4 gene	AF171708	Endoplasmic reticulum and Golgi (rats expression highest in brain)	[47,48]
S-adenosylmethionine decarboxylase gene	AF171712	Polyamine synthesis, presumably in every cell, highest in 24 and 48 h larvae	[49]
Twinstar gene	AF171714	Male germ line and larvae throughout development	[50,51]
CCATT box transmembrane domain gene	AF171715	Not specified	-
Geranylgeranyl transferase gene	AF171718	Not specified	-
ADP-robosylation factor class II gene	AF171722	Uniformly distributed ubiquitous in all adult body segments	[52,53]
Virus-like particle	AF171727	Long gland and ovipositor in adults	[54]
Rot gene	AF171731	Not specified	-
DNA-binding protein erect wing	AF171732	Throughout embryonic development and enriched in adult head	[55]
Membrane-associated protein gene	AF171736	Uniform in embryonic development	[56]
BBC-1 gene	AF171739	Not specified	-
Vimar gene	AF171743	Midgut and hindgut, visceral mesoderm, CNS, and PNS in embryos	[57]
Metallothionein gene	AF171741	Alimentary canal and lower in other tissues of larvae	[58]
Nicotinic acetylcholine receptor gene	AF171702	Brain and CNS predominantly in late embryos and adult head	[59,60,61]
Burdock retrotransposon gag protein gene	AF171716	Not specified	-
Transposable element to copia mgd3 retroposon	AF171724	Varies with Drosophila populations	[62,63]
	AF171725
Heat-shock gene hsp 27	AF171728	CNS, sperm, and oocytes, present in all stages, highest in white prepupae	[64,65]
Alpha 1,2 mannosidase gene	AF171730	Embryonic PNS, adult eye, and wing	[66]
GTP cyclohydrolase I	AF171733	Embryo nuclear, adult eye and head	[67,68]
Teashirt gene	AF171735	Epidermis and mesoderm during development	[69]
	AF171759
49 kD phosphoprotein	AF171737	Photoreceptors	[70]
	AF171738
Alcohol dehydrogenase related gene	AF171740	Not specified	-
Vacuolar-ATPase gene	AF171742	Uniform expression in all stages	[71]
Micropia-Dm11 3' flanking DNA	AF171746	Not specified	-
	AF171748
RM62 RNA helicase	AF171749	Not specified	-
ADP/ATP translocase	AF171704	Not specified	-
Ubiquitin protein gene	AF171707	Tissue-general, all life stages	[72]
Calmodulin gene	AF171711	CNS and mushroom bodies of adults	[73]
	AF171781
TRIP-1 homolog gene	AF171713	Not specified	-
	AF171726
Bnb gene for development	AF171729	Mesectoderm and presumptive epidermis, after dorsal closure periphery of nervous system including glia that may establish longitudinal neuropile scaffolding, embryonic CNS	[74]
	AF171751
B(2)gcn gene	AF171754	Not specified	-
Diacylglycerol kinase gene	AF171720	Eye-specific in adult nervous system, muscles, compound eye,	[75,76]
	AF171721	brain cortex, fibrillar muscle, and tubular muscle
	AF171755
	AF171756
Gene from heat-shock locus 93D	AF171760	Constitutive monitoring the 'health' of translation machinery, presumably in every cell	[77,78]
Cytochrome c oxidase gene	AF171709	Mitochondrial
	AF171710
	AF171719
	AF171723
	AF171734
	AF171752
BM40 gene	AF171872	Not specified	-
Histone H3.3 gene	AF171745	Gonads and somatic tissue, uniform distribution in polytene chromosomes	[79,80]
Acetylcholine receptor-related protein	AF171747	CNS	[81]
Hu-li tai shao gene	AF171750	Ovarian ring canal	[82]
Laminin receptor gene	AF171753	Neural tissue	[83]
eIF-2 alpha-subunit	AF171758	Expressed throughout embryos, and CNS in later stages	[84,85]
Gerceraldehyde-3-phosphate dehydrogenase-2 gene	AF171757	Evenly distributed, expressed in all stages	[86]
CNS-specific Noe gene	AF171772	CNS	[87]
	AF171796
	AF171818
	AF171848
	AF171780
Medea-B gene	AF171807	Not specified	-
Phospholipase C norpA gene	AF171840	Retina and body of adults	[88]
Recq helicase 5 gene	AF171784	DNA repair, recombination, and replication	[89]

Each previously identified gene is listed with its accession number from the Drosophila brain study. Location information is as reported by the indicated reference.

Approximately 42% of the sequence data generated in this study were originally novel according to sequence analysis searches conducted at the beginning of this project. Since then, much EST data has been added to GenBank and the Drosophila genome sequence has been released. Thus, in October 2000 the 114 previously novel brain cDNA were again compared with fly sequence data. The percentage of transcripts that do not have corresponding ESTs is reduced to 11% (Table 2; of 29 clones, 17 have no EST matches and are not predicted genes following genome annotation, and 12 have no EST matches but are matched with a predicted gene). Although each of these 29 clones lacks an EST match, each clone is identified within the Drosophila genome sequence recently reported by Adams et al. [35]. It is possible that some of these clones represent the 3' ends of ESTs for which only 5' sequence data is available. Considering that data for approximately 80,000 ESTs (24,193 ESTs from adult heads alone) are reported [36] and that our analysis examined only 271 randomly chosen brain library clones, 11% is a surprisingly large number. This indicates that this library is a valuable resource for generating sequence data that will facilitate genome annotation, specifically identifying regions transcribed in the adult fly brain.

From our analysis it is clear that EST data are essential for accurate and thorough genome annotation. In particular, using current genome annotation algorithms, 42 of the 271 brain clones do not correspond to predicted genes (Table 2). Of these 42 clones, however, 25 have EST matches with the Berkeley Drosophila Genome Project (BDGP) data (Tables 2, 5). Comparisons of the remaining 17 cDNA sequences with the Drosophila genome sequence show evidence of RNA processing (exon/intron borders and consensus splicing sequences) for two clones, and presence of a poly(A) addition sequence (AAUAAA) 12 to 30 bp upstream of an extensive poly(A) region at the 3' end of the insert sequence for seven clones (Table 5b). Ten of the 17 clones were detected in reverse northern experiments using either brain or body radiolabeled cDNA (Table 6). The distribution of detection by brain cDNA, body cDNA, both or neither (not detectable above background) for the 17 clones in this category is similar to the distributions observed in the other categories (Tables 5a, 6), and strikingly similar to the detection frequency observed for the 'matched with an EST and a predicted gene' category. Although these data suggest that these sequences are transcribed, additional experiments are necessary to confirm whether this is true for each clone. None of the clones in this category is predicted to encode a protein larger than 100 amino acids. It is possible that these sequences may correspond to genomic DNA. Alternatively, these novel RNA molecules may perform some unknown cellular function that requires a conserved structure rather than a conserved sequence.

Table 5a

Correlation between EST match, gene prediction and hybridization analysis

Detected in	Accession number	Insert size	Percentage of category
Novel cDNA
Body	AF171819	145	12%
Body	AF171858	186
Both	AF171764	450	12%
Both	AF171805	259
Brain	AF171789	857	35%
Brain	AF171794	610
Brain	AF171800	190
Brain	AF171808	269
Brain	AF171815	363
Brain	AF171854	195
Neither	AF171813	216	41%
Neither	AF171821	359
Neither	AF171828	189
Neither	AF171838	430
Neither	AF171850	1104
Neither	AF171859	1786
Neither	AF171865	452
Matched with an EST, but NOT a predicted gene
Body	AF171857	432	4%
Both	AF171772	480	32%
Both	AF171779	1101
Both	AF171787	449
Both	AF171804	553
Both	AF171818	180
Both	AF171839	1282
Both	AF171848	345
Both	AF171860	151
Brain	AF171766	798	36%
Brain	AF171780	292
Brain	AF171781	400
Brain	AF171782	506
Brain	AF171785	727
Brain	AF171796	313
Brain	AF171797	400
Brain	AF171811	386
Brain	AF171863	1072
Neither	AF171826	683	28%
Neither	AF171829	380
Neither	AF171837	503
Neither	AF171840	623
Neither	AF171845	376
Neither	AF171862	800
Neither	AF171869	641
Matched with a predicted gene, but NOT an EST
Body	AF171867	287	8.3%
Both	AF171768	1746	33.3%
Both	AF171778	389
Both	AF171790	396
Both	AF171799	819
Brain	AF171771	1060	33.3%
Brain	AF171783	373
Brain	AF171803	688
Brain	AF171812	338
Neither	AF171832	338	25%
Neither	AF171861	398
Neither	AF171868	689
Matched with an EST and a predicted gene
Body	AF171846	578	3%
Body	AF171856	600
Both	AF171763	236	30%
Both	AF171770	919
Both	AF171773	315
Both	AF171774	745
Both	AF171776	278
Both	AF171777	165
Both	AF171786	223
Both	AF171791	451
Both	AF171792	619
Both	AF171793	234
Both	AF171795	200
Both	AF171798	733
Both	AF171809	577
Both	AF171810	966
Both	AF171817	250
Both	AF171830	567
Both	AF179229	1396
Both	AF179230	168
Brain	AF171761	1081	27%
Brain	AF171762	652
Brain	AF171765	281
Brain	AF171767	228
Brain	AF171769	1051
Brain	AF171775	523
Brain	AF171788	1421
Brain	AF171801	600
Brain	AF171802	623
Brain	AF171806	727
Brain	AF171807	380
Brain	AF171814	237
Brain	AF171816	324
Brain	AF171822	376
Brain	AF171824	785
Brain	AF171833	868
Neither	AF171784	351	40%
Neither	AF171820	427
Neither	AF171823	234
Neither	AF171825	675
Neither	AF171827	253
Neither	AF171831	222
Neither	AF171834	240
Neither	AF171835	916
Neither	AF171836	406
Neither	AF171841	436
Neither	AF171842	331
Neither	AF171843	99
Neither	AF171844	764
Neither	AF171847	301
Neither	AF171849	193
Neither	AF171851	364
Neither	AF171852	364
Neither	AF171853	473
Neither	AF171855	991
Neither	AF171864	541
Neither	AF171866	412
Neither	AF171870	399
Neither	AF171871	488
Neither	AF171872	546

Hybridization results for the indicated cDNA categorized as 'novel cDNA'; 'matched with an EST, but not a predicted gene'; 'matched with a predicted gene, but not an EST'; or 'matched with an EST and a predicted gene' (see Table 2). Purified plasmids containing the indicated insert sequence were hybridized with either labeled brain or body cDNA (see Table 6). The percentage of clones exhibiting the indicated hybridization pattern within each category is indicated.

Table 6

Body versus brain expression of originally novel clones

Clone category	GenBank accession number	psl/mm2-bkg		Total
		Brain	Body
Brain only	AF171761	1.52	ND	34
	AF171765	0.68	ND
	AF171766	0.60	ND
	AF171767	1.62	ND
	AF171769	0.15	ND
	AF171771	0.79	ND
	AF171775	1.20	ND
	AF171780	4.18	ND
	AF171781	1.10	ND
	AF171782	1.03	ND
	AF171783	0.94	ND
	AF171785	1.52	ND
	AF171788	1.44	ND
	AF171789	0.77	ND
	AF171794	0.61	ND
	AF171796	3.74	ND
	AF171797	0.87	ND
	AF171800	1.01	ND
	AF171801	0.98	ND
	AF171802	0.46	ND
	AF171803	0.86	ND
	AF171806	1.15	ND
	AF171807	0.70	ND
	AF171808	0.92	ND
	AF171811	0.38	ND
	AF171812	0.60	ND
	AF171814	0.90	ND
	AF171815	0.91	ND
	AF171816	0.24	ND
	AF171822	5.43	ND
	AF171824	1.14	ND
	AF171833	0.34	ND
	AF171854	0.20	ND
	AF171863	3.73	ND
Body and brain	AF171762	3.94	0.01	33
	AF171763	2.81	8.92
	AF171764	1.13	0.06
	AF171768	1.76	0.39
	AF171770	1.25	0.70
	AF171772	14.1	0.81
	AF171773	1.03	0.43
	AF171774	0.77	0.04
	AF171776	1.09	0.43
	AF171777	1.20	1.65
	AF171778	1.45	2.48
	AF171779	0.67	0.55
	AF179229	2.46	0.09
	AF179230	1.38	3.88
	AF171786	0.77	0.03
	AF171787	1.14	1.02
	AF171790	1.17	0.80
	AF171791	1.07	6.13
	AF171792	1.25	1.56
	AF171793	1.19	1.47
	AF171795	3.32	0.68
	AF171798	0.92	0.31
	AF171799	1.30	0.05
	AF171804	1.05	0.47
	AF171805	1.11	0.13
	AF171809	2.22	0.49
	AF171810	1.34	0.12
	AF171817	0.63	0.01
	AF171818	18.3	0.94
	AF171830	0.31	0.01
	AF171839	9.93	2.40
	AF171848	20.7	0.13
	AF171860	23.4	0.67
Body only	AF171819	ND	12.9	6
	AF171846	ND	0.08
	AF171856	ND	0.06
	AF171857	ND	0.19
	AF171858	ND	0.35
	AF171867	ND	0.21
Neither body nor brain	AF171784	ND	ND	41
	AF171813	ND	ND
	AF171820	ND	ND
	AF171821	ND	ND
	AF171823	ND	ND
	AF171825	ND	ND
	AF171826	ND	ND
	AF171827	ND	ND
	AF171828	ND	ND
	AF171829	ND	ND
	AF171831	ND	ND
	AF171832	ND	ND
	AF171834	ND	ND
	AF171835	ND	ND
	AF171836	ND	ND
	AF171837	ND	ND
	AF171838	ND	ND
	AF171840	ND	ND
	AF171841	ND	ND
	AF171842	ND	ND
	AF171843	ND	ND
	AF171844	ND	ND
	AF171845	ND	ND
	AF171847	ND	ND
	AF171849	ND	ND
	AF171850	ND	ND
	AF171851	ND	ND
	AF171852	ND	ND
	AF171853	ND	ND
	AF171855	ND	ND
	AF171859	ND	ND
	AF171861	ND	ND
	AF171862	ND	ND
	AF171864	ND	ND
	AF171865	ND	ND
	AF171866	ND	ND
	AF171868	ND	ND
	AF171869	ND	ND
	AF171870	ND	ND
	AF171871	ND	ND
	AF171872	ND	ND

Purified plasmid DNA was spotted onto a nylon filter and hybridized with either radiolabeled brain or body cDNA. Clones are classified on the basis of whether they were detected with brain, body, both (brain and body) or neither (not above background) radiolabeled cDNA. The clones tested were originally novel (no EST or previous sequence information), but some clones have changed classification as a result of both the large number of ESTs submitted thorough the Drosophila genome effort (BDGP) and the Drosophila genome annotation efforts. Additional information for these clones is listed in Tables 2, 5. ND, not determined. Bkg, background.

The Drosophila genome is predicted to contain 13,601 genes [35]. Ifour observations are representative and can be extended to the number of genes in the fly genome, then our analysis suggests that the total number of genes may be underestimated by approximately 15% (42 of the 271 randomly chosen cDNAs do not correspond to a predicted gene). Thus, approximately 2,000 genes may await discovery.

Transcript distribution analysis

A second hybridization study was conducted to determine whether clones originally identified as novel were detectable in the brain and/or body of adult Drosophila. This data may offer clues as to which transcripts are involved in basic neuronal function, as opposed to a function that may be specific to the brain. The Drosophila central nervous system (CNS) includes thoracic and abdominal ganglia and, therefore, neural transcripts are often expressed throughout the body. Thus, it was possible that few transcripts would be brain-specific.

To determine how the (originally) novel clones were distributed in the animal, plasmid templates from 114 novel clones were spotted on filters and hybridized with radiolabeled cDNA from either brains or bodies (minus heads). The results of this study are listed in Table 6. In this experiment cDNA probe is limiting and, therefore, many transcripts that are in low abundance may not be detectable. In fact, 36% of the clones were not detected in either brain or body. These clones may correspond to less abundant transcripts. Ideally, hybridization probe would be in excess in these experiments to determine which clones are brain specific, but Drosophila brain cDNA is limiting. Approximately 30% of the clones were detectable only in the brain and are candidates for genes involved in brain function. Clones that were detected in both tissues made up about one third of the novel transcripts (29%). About 5% of the clones were detected only in the body. As the library is made from brain tissue, we did not expect to recover many transcripts that would only be detectable in the body, as compared to the brain.

We used published localization data from previously identified transcripts to evaluate the data we collected for the novel clones (Table 4). Approximately 22% (7 of 32) of the known Drosophila genes listed are neural-specific, and approximately 30% of novel transcripts were detected only in the brain. Approximately 29% of the novel transcripts were detectable in both brain and body tissues. Known Drosophila genes that were localized in body and brain tissues accounted for 56% (18 of 32) of genes for which localization data was available (Table 4). Clones detected in both tissues may indicate that the gene product is needed in all cells. Genes from the nervous system would be expected to be expressed in both tissues, so transcripts detected in both cannot be ruled out of this category; but these transcripts are not brain specific. Approximately 5% of the novel clones were detectable only in the body, as compared to 22% (7 of 32) of the known Drosophila clones detected only in body tissues. These transcripts are apparently expressed at a higher level in the body and at relatively low levels in the brain. It should be noted that localization data were not specified for 18 of the 49 (37%) known transcripts listed in Table 4. This analysis suggests that this brain cDNA library is a rich source for generating cDNA sequence information and for identifying novel, brain-specific cDNAs.

Conclusions

The initial analysis of an adult Drosophila brain library is presented here. Somewhat surprisingly, we observe no clear connection between the abundance of a transcript and its appearance in a sequence data bank. However, molecular screens that are directed towards isolating rare transcripts may skew the transcript-related data in sequence banks towards less abundant molecules. As shown in Figure 1 and Table 2, we have identified and sequenced 29 novel clones that do not match with other known expressed sequences (but do match with fly genomic sequence information), 85 clones that are matched with EST data, 71 clones that were previously reported Drosophila sequences, 39 clones that contain ribosomal protein sequences, 27 clones that are matched with genes previously reported for other organisms (isologs, Table 3) and 20 clones corresponding to mitochondrial sequences.

Why did we recover such a high percentage of novel sequences? Libraries made from brain tissue are proposed to have a higher complexity of transcripts than libraries made from other tissues [21]. Therefore, EST screens of brain libraries should yield larger numbers of independent transcripts, as a result of the increased transcript complexity within brain tissues. Another possible explanation for the surprisingly large number of novel cDNAs identified in our analysis is that our library is not normalized. It has been proposed that hybrids form between poly(dA) and poly(dT) sequences during the hybridization/subtraction reaction and that these sequences are subsequently lost [36].

An ultimate goal of this project is to create a database of all the transcripts expressed in the Drosophila brain and to correlate this information with their patterns of expression in the brain. This type of a database would be a valuable resource and could be used in comparative studies with other organisms. Comparisons of transcripts from organisms with relatively simple brains (Drosophila) to organisms with more complex neural function (humans) may offer insights into basic brain function and aid in the identification of transcripts involved in higher-order brain functions. The 35 clones that appear enriched in the brain may identify proteins or RNAs that are involved in a brain-specific function. Transcripts identified in this library can be directly tested for protein-protein interaction using the yeast two-hybrid capability of the library, making it a good resource for many areas of study.

Our analysis of this unique brain library demonstrates that many transcribed regions of the Drosophila genome remain undiscovered, and that approximately 2,000 more genes may be identified. Genome annotation efforts emphasize identifying protein-coding regions [37]. Thus, it is possible that some of the ESTs lacking a corresponding predicted gene were missed during genome annotation because an open reading frame (or one of sufficient size) was not predicted.

Complete genomic sequences are excellent resources, and extensive annotation of a genome makes the sequence information even more powerful. Current software is not sufficient to identify all transcribed regions within the genome. As of the year 2000, EST data for 24,193 clones from adult Drosophila head libraries is reported and estimated to represent over 40% of all Drosophila genes [38]. Our results confirm that not all transcribed regions of the genome are identified and that EST analyses are essential for accurate and complete genome annotation.

Materials and methods

Tissue preparation

To produce the animals for the brain dissections, adult Drosophila were entrained using 12 h light and 12 h darkness in temperature-controlled incubators at 25°C. Entrained adult D. melanogaster (Canton S) flies were collected 3 h after the lights were turned off, frozen on dry ice, shaken to detach heads from bodies, and separated through a screen to isolate the heads. Frozen heads were incubated in prechilled -20°C, 100% acetone (EM Science) at -20°C overnight to replace the water in the tissue with acetone [39]. Prechilling the acetone prevents the heads from thawing when added to the acetone. Heads were dried at room temperature, and brains were removed using fine dissecting tweezers.

RNA preparation

RNA was isolated according to the Micro RNA Isolation protocol from Stratagene, with the exception of homogenization. Dried tissue was homogenized in denaturing solution with β-mercaptoethanol for 1 min, incubated on ice for 15 min, and then homogenized for an additional 5 min. The addition of a rehydration step increased the yield of RNA from dried tissue to approximately the same level as fresh tissue (16-21.9 μg total RNA extracted from 100 fresh heads, 15-29.3 μg total RNA extracted from 100 acetone-dried heads with rehydration step, compared to 3-6.6 μg total RNA extracted from 100 acetone-dried heads with no rehydration step). Poly(A) RNA was isolated using the Poly(A) Quick mRNA Isolation Kit from Stratagene according to the manufacturer's instructions. Approximately 5 μg poly(A) RNA was extracted from approximately 15,000 brains. Weighing acetone-dried brains allowed us to estimate how many were used to construct the library (50 acetone-dried brains weigh 0.25 mg and 50 acetone-dried heads weigh 1.8 mg).

Library construction

The library was constructed using a Stratagene HybriZAP™ Library kit. First-strand cDNA synthesis was primed from the 3' end of the poly(A) RNA using a poly(T) primer that also contained an XhoI restriction site and a GAGA sequence (5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTITTTTTTTTTTT-3'). 5-methyl dCTP was used during first-strand cDNA synthesis to protect internal XhoI sites. Second-strand synthesis was primed by the partially digested RNA that resulted from RNase H treatment of the first-strand synthesis reaction. Pfu DNA polymerase was used to blunt the cDNA and EcoRI adapters were ligated to the blunt ends. The cDNA was digested with EcoRI and XhoI, size separated (retaining molecules approximately 200 bp or larger), ligated into HybriZAP™ vector arms, and packaged into phage heads for amplification.

Determining the number of primary clones

The cDNA library was titered to determine how many independent clones were recovered. At the 10-1 dilution there were 270 plaques per plate, giving a total of 6.75 × 106 clones in the primary library. This number was calculated as follows: (number of plaques 270) × (dilution factor 10) × (total packaging volume 500 μl) / (total number of mg packaged 8.75 × 10-5) × (number of μl packaged 1) = 1.542 × 1010 plaque-forming units (PFU) per mg or 1.35 × 106 PFU per packaging reaction. There are five packaging reactions for the entire library for a total of 6.75 × 106 clones in the primary library.

Sequencing-template preparation

PCR template

Individual phage plaques were incubated in 400 μl SM buffer overnight and used as amplification template. Amplification reactions were performed in a total volume of 40 μl and contained 2 μl eluted phage, 40 ng of each primer (FADI 5'-CACTACAATGGATGATG-3' and RADI 5'-CTTGCGGGGTTTTTCAG-3'), 0.001% Tween 20 (Sigma), 2.5 U Taq DNA polymerase (Promega), 1x Taq polymerase buffer (Promega), 1.56 mM MgCl2 (Promega), and 0.25 mM of each dNTP (USB). PCR was performed on a Perkin-Elmer 9600 GeneAmp PCR system, as specified in the HybriZAP™ Two-Hybrid cDNA Gigapack Cloning Kit instruction manual (Stratagene). After amplification, reactions were incubated at 37°C for 15 min with 0.5 Uμl-1 exonuclease I (USB) and 0.5 Uμl-1 shrimp alkaline phosphatase (Amersham Life Sciences). Enzymes were inactivated by heat treatment at 85°C for 15 min. Resulting samples were electrophoretically separated on a 1% Agarose (Kodak) gel and compared to a quantitative marker, BioMarker-EXT (BioVentures), to estimate the DNA concentration of each sample. This DNA was directly used in subsequent sequencing reactions.

Plasmid template

Individual phage plaques were incubated in 400 μl SM buffer overnight and then used for excision. Library phage were incubated with ExAssist Helper Phage™ (Stratagene) and XL1-Blue Escherichia coli cells, and grown overnight in Luria Broth. E. coli cells were killed by heat treatment (70°C, 20 min). XLOR E. coli cells were inoculated with the released phagemids and this mixture was plated on 50μg ml-1 ampicillin (Sigma) selection medium. Resulting colonies were cultured for subsequent plasmid DNA preparation (Perfect Prep Plasmid DNA kit, 5'-3' Inc.).

Sequencing

Initial sequence information was obtained using standard sequencing methods (described below) and a vector primer directed toward the 5' end of the insert (FADI 5'-CACTACAATGGATGATG). These ESTs were evaluated using the BLAST search program [40,41] linked to the nonredundant GenBank database. Novel cDNAs and isologs were completely sequenced. If a cDNA had been previously identified, sequence determination was not continued. The second standard sequencing reaction was primed from poly(A) tail using 1.6 pmol of a poly(T) primer anchored (PLYT 5'-TTTTTTTTTTTTTTTV-3' (V=A, C, or G)). cDNA sequences were completed using an octamer-primer walking strategy [42,43].

Automated sequencing reactions were performed using ABI PRISM Dye Terminator (or dRhodomine for PLYT reactions) Cycle Sequencing Ready Reaction Kits with AmpliTaq DNA polymerase, FS, according to the manufacturer's directions or as described for octamer-primed sequencing reactions [42,43]. The FADI primer was annealed at 48°C and the PLYT primer was annealed at 20°C. Sequencing reactions were ethanol precipitated, pellets were resuspended in 3.5 μl loading buffer, 1.5 μl was loaded onto a sequencing gel, and the data was collected by an ABI PRISM 377 DNA sequencer. Data collected from the ABI PRISM 377 DNA sequencer was manually edited using Sequencher 3.0 (GeneCodes).

Hybridization analyses

Eighty-five individual phage plaques were incubated in 400 μl SM buffer overnight and 2 μl phage eluant was used to produce a grid of plaques on a lawn of E. coli cells. Filter lifts were taken from the grid of plaques and hybridized at 65°C overnight with labeled Drosophila head cDNA at 1 × 106 cpm per ml hybridization buffer (50% formamide, 5x SSC, 0.1% Ficoll w/v, 0.1% PVP w/v, 0.1% BSA w/v, 0.1% SDS w/v, 0.2 μg ml-1 salmon sperm DNA and 1 mM EDTA). Filters were then washed sequentially at 42°C in 5x SSC for 1 h, at 65°C in 1x SSC for 1 h, and at 65°C in 0.1x SSC for 1 h. Filter lifts were exposed to phosphorimaging plates for 24 h (Fuji Medical Systems) and psl (photo stimulating units) of a standard area were determined using a Fuji Bas1000 Imager.

Hybridization of all novel clones

Plasmid DNA (10 ng) from each novel clone was denatured at 95°C for 5 min and spotted onto a nylon filter. Filters were hybridized at 65°C overnight with either labeled Drosophila body (minus head) or labeled Drosophila brain cDNA at 1 × 106 cpm ml-1 in Church and Gilbert buffer (7% SDS, 1 mM EDTA, 500 mM Na2HPO4, and pH to 7.2 with H3PO4 [44]). Filters were washed twice for 1 h (each) at 65°C in Church and Gilbert buffer. Filters were then exposed to phosphorimaging plates for 24 h, and psl of a standard area was determined using a Fuji Bas1000 Imager. 10 ng of each plasmid on the filter contains approximately 1.2 × 1012 copies and, therefore, probe is expected to be limiting in these experiments.

Categorizing clones

Clones classified as 'novel' had no obvious match to nucleic acid/protein sequence information in GenBank (a score less than 100). Novel clones may contain blocks of less than 100 bases that are matched with other sequences, but these small regions have no known functional correlation and the similarity between the two sequences was very low. It is possible that some of our novel clones could be part of an EST from the BDGP that has not been fully sequenced. Sequences categorized as matched to EST data have a high degree of similarity (a score over 100) to reported sequence information, but the previously collected sequence data was not associated with a known function. Sequences categorized as 'known Drosophila' were a perfect match (with perhaps the exception of a few bases, fewer than 10) with sequence information from Drosophila. 'Matched isologs' are sequences that have a high degree of similarity (score of over 100 at the protein level) to a gene found in another organism, but a functional homology between these genes has not been determined. Ribosomal protein sequences were categorized as 'known' or 'isologs' using the above criteria. Ribosomal protein and mitochondrial sequence clones were categorized separately because these types of transcripts frequently occurred in our library.