Insect Neuropeptides - American Chemical Society

Chapter 20 Adipokinetic Hormone Neuropeptide Family Applying Recombinant DNA Techniques Martin H. Schaffer and Barbara E. Noyes

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch020

Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75240-9070

The application of recombinant DNA techniques to the study of neuropeptide biosynthesis is essential because these techniques facilitate structural analyses and evolutionary studies, c l a r i f y biosynthetic pathways, provide the necessary background and probes for analysis of synthesis regulation at the l e v e l s of mRNA and gene transcription and assist in classical genetic experimentation. Examples of these benefits are cited and problems encountered in insect systems are discussed. The a n a l y s i s o f n e u r o p e p t i d e s i n i n s e c t s may l e a d t o t h e development o f b o t h s p e c i f i c i n s e c t c o n t r o l s t r a t e g i e s and model systems t h a t c l a r i f y t h e r o l e o f n e u r o p e p t i d e s i n a l l a n i m a l s . T h e r e f o r e i t i s i m p o r t a n t t o v i g o r o u s l y pursue n e u r o p e p t i d e s t u d i e s and t o e x p l o i t t h e a v a i l a b l e t e c h n o l o g i e s t o t h e f u l l e s t . This p a p e r w i l l r e v i e w t h e i m p o r t a n c e o f c u r r e n t r e c o m b i n a n t DNA t e c h n i q u e s t o t h e study o f n e u r o p e p t i d e b i o l o g y . S t u d i e s o f t h e a d i p o k i n e t i c hormone (AKH) f a m i l y w i l l be emphasized as examples, but o t h e r p e p t i d e s w i l l be mentioned t o p r e s e n t a broader p i c t u r e of the advantages and problems a s s o c i a t e d w i t h the t e c h n i q u e s . There a r e s e v e r a l g e n e r a l reasons f o r s t u d y i n g n e u r o p e p t i d e r e l a t e d mRNAs and genes i n c l u d i n g : I. E f f i c i e n t d e t e r m i n a t i o n o f p r i m a r y sequences I I . D e t e r m i n a t i o n o f b i o s y n t h e t i c pathways I I I . A n a l y s i s o f the r e g u l a t i o n o f s y n t h e s i s IV. F a c i l i t a t i o n o f g e n e t i c s t r a t e g i e s t o s t u d y f u n c t i o n V. C l a r i f i c a t i o n of evolutionary relationships and these w i l l be c o n s i d e r e d i n t u r n . P r i m a r y Sequences One o f the most o b v i o u s b e n e f i t s o f modern DNA t e c h n o l o g y i s t h e e x p l o s i o n i n a v a i l a b l e s t r u c t u r a l d a t a as a r e s u l t o f the r e l a t i v e 0097-6156/91/0453-0226$06.00/0 © 1991 American Chemical Society Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


20. SCHAFFER AND NOYES

Adipokinetic Hormone Neuropeptide Family

ease o f DNA sequencing. T h i s technology i s p a r t i c u l a r l y u s e f u l i n c e r t a i n s p e c i a l c a s e s . For example the d e t e r m i n a t i o n o f l o n g amino a c i d s e q u e n c e s i s u s u a l l y s i m p l e r a t t h e DNA t h a n t h e p r o t e i n l e v e l . W h i l e t h i s i s t y p i c a l l y not a problem w i t h n e u r o p e p t i d e s i t w i l l become i m p o r t a n t as i n v e s t i g a t o r s b e g i n t o s t u d y the r e c e p t o r m o l e c u l e s t o which n e u r o p e p t i d e s (and p o t e n t i a l c o n t r o l agents) a r e t a r g e t e d . There w i l l a l s o be i n s t a n c e s i n which a n e u r o p e p t i d e i s too l a r g e t o be sequenced c o m p l e t e l y from i t s amino t e r m i n u s , and s o s c a r c e t h a t e x t e n s i v e s t r u c t u r a l s t u d i e s s u c h as c o m p l e t e t r y p t i c mapping a r e u n f e a s i b l e . I n t h i s i n s t a n c e , amino t e r m i n a l d a t a can p r o v i d e a b a s i s f o r complete s t r u c t u r a l d e t e r m i n a t i o n by cDNA cloning. Recombinant DNA t e c h n i q u e s have been p a r t i c u l a r l y u s e f u l i n p r o v i d i n g t h e p r i m a r y amino a c i d s e q u e n c e s o f neuropeptide p r e c u r s o r p r o t e i n s . These p r o t e i n s v a r y i n s i z e , but a r e sometimes quite large. S i n c e p r e c u r s o r s f u n c t i o n as m e t a b o l i c i n t e r m e d i a t e s they i n v a r i a b l y are found i n v e r y low abundance i f they a r e d e t e c t a b l e a t a l l . Thus i t i s u s u a l l y n e c e s s a r y t o use recombinant DNA t e c h n i q u e s t o d e t e r m i n e t h e a m i n o a c i d s e q u e n c e o f a n e u r o p e p t i d e p r e c u r s o r , and f o r t u n a t e l y a s e a r c h of recombinant l i b r a r i e s c a n u s u a l l y be b a s e d on t h e s t r u c t u r a l i n f o r m a t i o n p r o v i d e d by the n e u r o p e p t i d e i t s e l f . The importance o f n u c l e i c a c i d approaches i s i l l u s t r a t e d by the case o f Drosophila FMRFamiderelated peptides (1,2). The p r e c u r s o r i s l a r g e and t h e r e i s no r e c o g n i z e d r i c h s o u r c e o f the p e p t i d e s (much l e s s t h e i r p r e c u r s o r ) so d i r e c t s t r u c t u r a l d e t e r m i n a t i o n a t the p r o t e i n l e v e l would have been v e r y d i f f i c u l t indeed. The grasshopper AKH p e p t i d e s , on the o t h e r hand, were e x c e p t i o n a l i n t h a t the p r e c u r s o r p r o t e i n s ( l e s s t h e i r s i g n a l s e q u e n c e s ) w e r e i d e n t i f i e d and s e q u e n c e d a t t h e p r o t e i n l e v e l thanks t o i n t e n s e s y n t h e s i s i n the c o r p o r a c a r d i a c a ( C C . ) and t h e d i l i g e n c e o f H e k i m i e t a l . (3). Even i n t h i s n e a r l y i d e a l c a s e , as we s h a l l see, the s t r u c t u r e s o f the mRNAs p r o v e t o be v e r y u s e f u l (4,5). The p r e c u r s o r s f o r t h e g r a s s h o p p e r AKHs, Manduca AKH, the Drosophila p e p t i d e s r e l a t e d t o FMRFamide, and d r o s u l f a k i n i n a l l contain additional peptides. Thus the d e t e r m i n a t i o n of n e u r o p e p t i d e p r e c u r s o r s t r u c t u r e o f t e n pays the d i v i d e n d o f l e a d i n g t o the i d e n t i f i c a t i o n o f o t h e r i n t e r e s t i n g p e p t i d e s . I f one g r a n t s t h a t recombinant DNA t e c h n i q u e s have u t i l i t y i n the d e t e r m i n a t i o n o f new sequences, when can they be e x p l o i t e d ? The answer i s whenever two fundamental t e c h n i c a l problems, how t o o b t a i n a l i b r a r y c o n t a i n i n g the recombinant o f i n t e r e s t and how t o o b t a i n a probe s u i t a b l e f o r s c r e e n i n g the l i b r a r y , can be s o l v e d . The most o b v i o u s p l a c e t o b e g i n a c l o n i n g p r o j e c t i s o f t e n w i t h the c o n s t r u c t i o n o f an a p p r o p r i a t e cDNA l i b r a r y . T h i s i s u s u a l l y much more c h a l l e n g i n g than the c o n s t r u c t i o n o f genomic l i b r a r i e s , and a number o f d i f f i c u l t i e s may a r i s e . S c a r c i t y o f s t a r t i n g m a t e r i a l i s a common problem i n i n s e c t b i o c h e m i s t r y , and i t c e r t a i n l y was a p r o b l e m i n t h e c a s e o f c l o n i n g o f t h e S c h i s t o c e r c a n i t a n s AKH cDNAs. A l t h o u g h the C C . i s an abundant source o f the p e p t i d e s i t i s a l s o a t i n y s t r u c t u r e , and i t was n e c e s s a r y t o c o l l e c t n e a r l y 2000 organs i n o r d e r t o o b t a i n a manageable l e v e l o f p o l y ( A ) RNA a t t h e end o f t h e p r e p a r a t i o n . E v e n g i v e n a d e q u a t e amounts o f s t a r t i n g m a t e r i a l , t h e p r e p a r a t i o n o f h i g h q u a l i t y RNA i s n o t +

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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n e c e s s a r i l y assured. Techniques w h i c h work i n one t i s s u e i n one organism may be s p e c t a c u l a r l y u n s u c c e s s f u l i n another s e t t i n g . F o r example the g u a n i d i n e t h i o c y a n a t e e x t r a c t i o n p r o c e d u r e w h i c h worked w e l l f o r us i n p r e v i o u s work (6,7) gave low y i e l d s when a p p l i e d t o Schistocerca nitans C.C., w h i l e m o d i f i c a t i o n s o f another t e c h n i q u e proved e n t i r e l y s a t i s f a c t o r y (S). Researchers studying i n s e c t s s h o u l d be m i n d f u l o f t h e f a c t t h a t they a r e l i k e l y t o be i n t h e p o s i t i o n o f e x t r a c t i n g RNA from a n o v e l t i s s u e o r i n a s p e c i e s n o t previously studied. There i s a another s e t o f problems which may o c c u r d u r i n g t h e c o n s t r u c t i o n o f a cDNA l i b r a r y . F o r e x a m p l e , some RNAs a r e d i f f i c u l t t o c o n v e r t i n t o cDNAs. T h i s was t o some e x t e n t t r u e o f AKH I , w h i c h i s p r e d i c t e d by computer m o d e l i n g t o have a s u b s t a n t i a l amount o f s e c o n d a r y s t r u c t u r e (J5jrJ3) . R a t c h o l e c y s t o k i n i n mRNA a l s o proved t o be a temperamental t e m p l a t e and the cDNA c l o n e s d e r i v e d from i t were s h o r t by over 100 n u c l e o t i d e s (10) . Such problems c a n form a s i g n i f i c a n t b a r r i e r t o b e g i n n i n g a new p r o j e c t , s i n c e i t i s p o s s i b l e t h a t t h e s h o r t segment o f t h e mRNA c o d i n g f o r a known n e u r o p e p t i d e i s i n a c c e s s i b l e t o r e v e r s e transcription. However, once a l i b r a r y i s c o n s t r u c t e d , i t i s a v a i l a b l e as a r e s o u r c e f o r many i n v e s t i g a t o r s w i t h many i n t e r e s t s , and s c r e e n i n g a n e x i s t i n g l i b r a r y r e q u i r e s o n l y a m o d e s t e x p e n d i t u r e o f time and r e s o u r c e s . I t i s a l s o l i k e l y that as e x p e r i e n c e i n t h e f i e l d a c c u m u l a t e s c u r r e n t p r o b l e m s w i l l be a d d r e s s e d more e a s i l y . F o r e x a m p l e , i n some c a s e s i t may be p o s s i b l e t o a v o i d t h e c o n s t r u c t i o n o f l i b r a r i e s from a number o f c l o s e l y r e l a t e d s p e c i e s by u s i n g known n u c l e o t i d e sequence d a t a from one s p e c i e s and t h e t e c h n i q u e o f polymerase c h a i n r e a c t i o n (11) t o g e n e r a t e the r e l a t e d recombinants from another s p e c i e s . The p r o b l e m o f o b t a i n i n g a cDNA l i b r a r y c a n s o m e t i m e s be a v o i d e d , a t l e a s t i n i t i a l l y , by b e g i n n i n g a t t h e genomic l e v e l . Thus i n t h e c a s e s o f t h e d r o s u l f a k i n i n and t h e FMRFamide r e l a t e d p e p t i d e s o f Drosophila p r e c u r s o r s , investigators exploited the s m a l l Drosophila genome, and began t h e i r s c r e e n s a t t h i s l e v e l (1,2,12) . I n t h e s e c a s e s t h e a b s e n c e o f a r i c h s o u r c e o f mRNA meant t h a t cDNA l i b r a r i e s would be l e s s p r e d i c t a b l e and l i k e l y more d i f f i c u l t t o screen. I t i s important t o r e a l i z e that t h i s s i t u a t i o n does not a p p l y t o a l l i n s e c t s . A c r i d i d g r a s s h o p p e r s , f o r example, have genomes s e v e r a l t i m e s t h e s i z e o f mammals and thus n e a r l y 100 t i m e s the s i z e o f Drosophila (13). T h i s means t h a t t h e s c r e e n i n g o f a grasshopper genomic l i b r a r y r e q u i r e s a v e r y a c c u r a t e probe c a p a b l e o f a v o i d i n g t h e l a r g e number o f chance matches t h a t a r i s e i n such a l a r g e c o l l e c t i o n o f sequences, and t h a t a l a r g e number o f r e c o m b i n a n t s m u s t be s c r e e n e d . F o r example, a t t h e b e g i n n i n g o f o u r s e a r c h f o r t h e Schistocerca nitans AKH genes we e s t i m a t e d t h a t we would have t o s c r e e n over 1 m i l l i o n c l o n e s t o have a 90% chance o f e n c o u n t e r i n g a t r u e p o s i t i v e . And i t appears t h a t t h i s was a r e a s o n a b l y a c c u r a t e e s t i m a t e . The o t h e r t o o l n e c e s s a r y f o r a s u c c e s s f u l c l o n i n g p r o j e c t i s a probe w i t h s p e c i f i c i t y adequate f o r t h e l i b r a r y o f i n t e r e s t . The s p e c i f i c i t y r e q u i r e d w i l l v a r y g r e a t l y depending on t h e c o m p l e x i t y o f the l i b r a r y . Most o f t e n , c l o n i n g p r o j e c t s b e g i n w i t h the use o f s y n t h e t i c o l i g o n u c l e o t i d e s whose sequences a r e based on a v a i l a b l e p r o t e i n sequence d a t a . I n f a c t t h i s t e c h n i q u e has been employed i n





t h e c a s e s o f AKH I a n d I I , M a n d u c a AKH, d r o s u l f a k i n i n , a n d t h e Drosophila FMRFamide p r e c u r s o r (1,2,4,5,12,14) o f t e n , however, w i t h s p e c i a l m o d i f i c a t i o n s . F o r example, i n t h e case o f AKH I and I I , s p e c i f i c mRNA p r i m i n g was used t o improve t h e s p e c i f i c i t y o f the probes (5,15). Probe s p e c i f i c i t y i s o f t e n a problem i n p r o j e c t s based on s h o r t n e u r o p e p t i d e sequences. B r a d f i e l d and K e e l e y (14) d e a l t w i t h t h e s i m i l a r d i f f i c u l t i e s i n t h e Manduca AKH sequence through the use o f o l i g o n u c l e o t i d e s c o n t a i n i n g d e o x y i n o s i n e . W h i l e they were s u c c e s s f u l , s c r e e n i n g r e q u i r e d v e r y c a r e f u l l y a d j u s t e d c o n d i t i o n s , and i t was t h e i r c o n c l u s i o n t h a t t h e modestly s i z e d Manduca sexta genome s t r e t c h e d t h e power o f t h e technique t o i t s l i m i t . On t h e o t h e r hand two groups have o b t a i n e d p r e c u r s o r c l o n e s w i t h o u t t h e b e n e f i t o f any p r o t e i n d a t a from t h e s p e c i e s under investigation. S c h n e i d e r and T a g h e r t {2) c o m b i n e d t h e u s e o f o l i g o n u c l e o t i d e s d i r e c t e d a t t h e m o l l u s c a n p e p t i d e FMRFamide t o g e t h e r w i t h a cDNA c l o n e o f t h e Aplysia p r e c u r s o r t o o b t a i n a r e l a t e d gene i n Drosophila. N i c h o l s e t a l . (12) based a s e a r c h f o r a Drosophila p e p t i d e r e l a t e d t o g a s t r i n and c h o l e c y s t o k i n i n p r i m a r i l y on t h e p r o t e i n s e q u e n c e o f t h e c o c k r o a c h peptide leucosulfakinin (16) . I n both cases the s m a l l s i z e of the D r o s o p h i l a genome e x p e d i t e d t h e s e a r c h . U n f o r t u n a t e l y such s t r a t e g i e s w i l l n o t always be s u c c e s s f u l . F o r example comparison of t h e Schistocerca nitans AKH I and I I p r e c u r s o r s ' n u c l e o t i d e sequences w i t h t h a t o f Manduca AKH shows no s i g n i f i c a n t homology. Thus a cDNA o f a p r e c u r s o r f o r one o f t h e s e AKHs w o u l d n o t be u s e f u l i n s c r e e n i n g a l i b r a r y from the other s p e c i e s . I t a l s o s h o u l d be kept i n mind t h a t s u c c e s s o f such a s t r a t e g y does n o t o b v i a t e t h e need f o r s t r u c t u r a l s t u d i e s a t t h e p r o t e i n l e v e l . N o n e t h e l e s s , when s u c c e s s f u l , t h i s k i n d o f a s t r a t e g y can r a p i d l y advance t h e a r e a under s t u d y . A n t i b o d i e s c a n a l s o be used as probes t o s c r e e n e x p r e s s i o n l i b r a r i e s , and t h i s s t r a t e g y has been used s u c c e s s f u l l y i n t h e c l o n i n g of v e r t e b r a t e neuropeptides (17). I t i s l i k e l y that this t e c h n i q u e w i l l be h e l p f u l i n cases i n which s t r u c t u r a l d a t a i s p a r t i c u l a r l y d i f f i c u l t t o o b t a i n and a v a r i e t y o f h i g h l y s p e c i f i c antibodies i s available. W h i l e these and o t h e r t e c h n i q u e s w i l l prove v a l u a b l e i n s p e c i a l c i r c u m s t a n c e s , s c r e e n i n g w i t h s y n t h e t i c o l i g o n u c l e o t i d e probes based on p r o t e i n d a t a i s l i k e l y t o be t h e p r i n c i p a l method o f i d e n t i f y i n g o t h e r p e p t i d e p r e c u r s o r s i n t h e near f u t u r e . D e t e r m i n a t i o n o f S y n t h e t i c Pathways D e t e r m i n i n g t h e s t r u c t u r e o f a cDNA n o t o n l y p r o v i d e s s t r u c t u r e s o f f i n a l p r o d u c t s , b u t a l s o r e v e a l s much about how those p r o d u c t s a r e formed. F o r example, i t was p o s s i b l e t o sequence t h e proAKH I and I I p e p t i d e s , b u t i t would n o t have been o b v i o u s whether t h e r e were l a r g e r v e r y s h o r t l i v e d p r e c u r s o r s t o these r a t h e r s m a l l p e p t i d e s w i t h o u t comparison t o t h e s t r u c t u r e s d e r i v e d from t h e mRNAs. Thus i t was i m p o r t a n t t o f i r s t a c c u r a t e l y e s t a b l i s h t h e completeness o f t h e cDNA c l o n e s and then i n s p e c t t h e i n f e r r e d p r e c u r s o r p r o t e i n s t r u c t u r e s . Both p r e c u r s o r s were found t o c o n t a i n a d d i t i o n a l amino t e r m i n a l sequences w h i c h were v e r y hydrophobic and l i k e l y t o be


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ACTION

s i g n a l sequences. The von H e i n j e a l g o r i t h m (18) p r e d i c t s c l e a v a g e j u s t p r i o r t o t h e G i n w h i c h b e g i n s b o t h m a t u r e AKH I and the i s o l a t e d proAKH I dimer ( i n the form of p G l u ) . This algorithm p r e d i c t s c l e a v a g e one amino a c i d c l o s e r t o the amino t e r m i n u s i n the case of AKH I I , but the a l g o r i t h m makes t h i s e r r o r f a i r l y o f t e n (20% of c a s e s ) . Such " p r e p r o " sequences are g e n e r a l l y v e r y u s e f u l i n p r e d i c t i n g t h e g e n e r a l scheme o f s y n t h e s i s , b u t i t i s n o t p o s s i b l e t o d e c i d e which p o t e n t i a l p r o c e s s i n g s i t e s are a c t u a l l y used. For example, both proAKH I and I I c o n t a i n the sequence a r g l y s i n t h e i r c a r b o x y l t e r m i n a l p e p t i d e s which a p p a r e n t l y are not p r o c e s s e d i n the C C . a t l e a s t . S i m i l a r l y i t would not be p o s s i b l e t o p r e d i c t a p r i o r i t h a t the s i n g l e c y s t e i n e s i n t h e s e p r o p e p t i d e s would o x i d i z e t o form p e p t i d e dimers (3,19). N o n e t h e l e s s the i n f e r r e d p r o t e i n sequence s e t s the b o u n d a r i e s f o r what i s p o s s i b l e , and o f t e n p r e d i c t s the e x i s t e n c e of u n r e c o g n i z e d p e p t i d e s w h i c h s h o u l d be s o u g h t i n t h e a n i m a l , s u c h as t h e many i n t e r e s t i n g p e p t i d e s i n the Drosophila FMRFamide r e l a t e d p e p t i d e precursor (1,2). Regulation

of

Synthesis

A s t r i k i n g f e a t u r e of n e u r o t r a n s m i t t e r c h e m i s t r y i s the p r e c i s e r e g u l a t i o n o f s y n t h e s i s , t h a t , f o r e x a m p l e , r e s u l t s i n a few neurons s y n t h e s i z i n g a p a r t i c u l a r n e u r o p e p t i d e w h i l e a l l the r e s t do n o t . I t appears from the v e r t e b r a t e l i t e r a t u r e t h a t mature neurons a l s o p r e c i s e l y r e g u l a t e the r a t e o f n e u r o p e p t i d e s y n t h e s i s ( 2 0 ) , though the f u n c t i o n a l s i g n i f i c a n c e of t h i s r e g u l a t i o n has not y e t been demonstrated, and i t seems p r o b a b l e t h a t i n s e c t s w i l l show t h i s phenomenon as w e l l . I t i s l i k e l y t h a t much of t h i s r e g u l a t i o n w i l l be a t the l e v e l of t r a n s c r i p t i o n , and so an u n d e r s t a n d i n g o f the phenomena of i n t e r e s t w i l l r e q u i r e a n a l y s i s o f both mRNA and gene s t r u c t u r e s as w e l l as the i d e n t i f i c a t i o n o f r e g u l a t o r y s i t e s and f a c t o r s . W h i l e such a n a l y s e s are j u s t b e g i n n i n g i n s e v e r a l i n s e c t n e u r o p e p t i d e s y s t e m s , t h e r e s u l t s w i l l be o f general i n t e r e s t because of the power of s i m p l e r n e u r a l systems t o c l a r i f y f u n c t i o n a l consequences of n e u r a l c e l l b i o l o g y . Genetic

Studies

B i o l o g i s t s and b i o c h e m i s t s i n t e r e s t e d i n i n s e c t s b e n e f i t from the f a c t s t h a t Drosophila i s such a f a v o r a b l e organism f o r g e n e t i c r e s e a r c h and has s u c h a r i c h h i s t o r y o f p r e v i o u s s t u d i e s . P a r t i c u l a r l y b e c a u s e g e n e t i c s t u d i e s h a v e s u c h power i n t h e a n a l y s i s of complex b i o l o g i c a l phenomena, i t seems i m p o r t a n t t o use g e n e t i c t e c h n i q u e s t o c l a r i f y the r o l e of n e u r o p e p t i d e s and the r e g u l a t i o n of t h e i r s y n t h e s i s t o the f u n c t i o n i n g o f the i n s e c t nervous system. One might a l s o imagine u s i n g mutants t o c l a r i f y the p o t e n t i a l of c o n t r o l agents d e s i g n e d t o e i t h e r b l o c k or over stimulate neuropeptide receptors. I t i s thus not s u r p r i s i n g t h a t s e v e r a l groups have begun the study of n e u r o p e p t i d e p r e c u r s o r s i n Drosophila and c e r t a i n l y more w i l l f o l l o w . We have j o i n e d i n by r e c e n t l y i s o l a t i n g an AKH p e p t i d e f a m i l y member from Drosophila ( S c h a f f e r , M.H.; N o y e s , B.E.; S l a u g h t e r , C.A.; Thome, G.C; G a s k e l l , S.J. Biochem. J . , i n p r e s s . ) , and u s i n g t h i s sequence t o



20.

SCHAFFER AND

NOYES


i d e n t i f y a genomic c l o n e . However, t h e c o m p l e x i t y of g e n e t i c s t u d i e s s h o u l d not be u n d e r e s t i m a t e d . D e s p i t e the l o n g h i s t o r y o f Drosophila g e n e t i c r e s e a r c h , i t i s l i k e l y t h a t i n f o r m a t i v e mutants and d e f i c i e n c i e s w i l l need t o be newly c o n s t r u c t e d , as i n the case o f D r o s o p h i l a FMRFamide (1,2). The work may w e l l be f u r t h e r c o m p l i c a t e d by the p o s s i b i l i t y of the mutants h a v i n g v e r y s u b t l e phenotypes. Even so an i m p r e s s i v e a r r a y o f t o o l s i s a v a i l a b l e t o a d d r e s s t h e s e p r o b l e m s , and t h e a d v a n t a g e s o f a d d i n g g e n e t i c approaches t o the o t h e r ways o f s t u d y i n g n e u r o p e p t i d e function j u s t i f y the c o n s i d e r a b l e e f f o r t i t i s l i k e l y t o r e q u i r e . W h i l e Drosophila i s c e r t a i n l y the i n s e c t w h i c h has been most i n t e n s i v e l y s t u d i e d , i t i s worth m e n t i o n i n g t h a t g e n e t i c s t u d i e s may become i m p o r t a n t i n o t h e r genera as we s t r u g g l e w i t h problems o f i n s e c t i c i d e r e s i s t a n c e , f o r example. F o r t u n a t e l y , o t h e r i n s e c t s do have some o f the f e a t u r e s which make Drosophila f a v o r a b l e f o r s t u d y , such as p o l y t e n e chromosomes, and i t may be t h a t recombinant DNA r e s e a r c h combined w i t h such f e a t u r e s w i l l encourage much more work i n o t h e r s p e c i e s . Evolutionary Studies I t seems o n l y common s e n s e t h a t t h e s t u d y o f g e n e s and t h e i r s t r u c t u r e s h o u l d be i n v a l u a b l e t o t h e s t u d y o f e v o l u t i o n , and indeed t h a t i s the case. One can a l r e a d y observe some o f these b e n e f i t s i n the work done on n e u r o p e p t i d e s . The d e t e r m i n a t i o n o f p r e c u r s o r s t r u c t u r e i s u s e f u l i n i d e n t i f y i n g or confirming evolutionary relations. A l t h o u g h the r e l a t i o n s h i p between AKH I and I I and Manduca AKH was a l r e a d y q u i t e s t r i k i n g , i t i s the case t h a t the Manduca AKH i s one o f the l e a s t t y p i c a l f a m i l y members (being 9 amino a c i d s i n l e n g t h i n s t e a d o f 8 o r 10) (21) , and i t i s p o s s i b l e t h a t s u c h a s t r u c t u r e m i g h t a r i s e due t o c o n v e r g e n t evolution. T h i s p o s s i b i l i t y i s argued a g a i n s t by the o b s e r v a t i o n t h a t the p r e c u r s o r p e p t i d e s show the same g e n e r a l o r g a n i z a t i o n and t h a t each o f these p r e c u r s o r s b e a r s a s i g n i f i c a n t l y h i g h e r degree of homology toward the o t h e r two than t o any o t h e r sequence i n the p r o t e i n sequence d a t a banks (4,5,14). The Drosophila FMRFamide r e l a t e d p r e c u r s o r p r o v i d e s a more s t r i k i n g example. A l t h o u g h o n l y one of the p e p t i d e s p r e d i c t e d by the i n f e r e d p r e c u r s o r s t r u c t u r e has been i s o l a t e d from the a n i m a l , o t h e r s w i l l d o u b t l e s s l y f o l l o w , and i t i s easy t o imagine t h a t the r e l a t i o n s h i p between some o f the more d i v e r g e n t phe-amide p e p t i d e s would be cause f o r s p e c u l a t i o n and debate i n the absence o f the p r e c u r s o r s t r u c t u r e . I n g e n e r a l , i t i s d i f f i c u l t t o be c o n f i d e n t about the e v o l u t i o n a r y s i g n i f i c a n c e o f homologies i n s h o r t n e u r o p e p t i d e s and the a n a l y s i s of p r e c u r s o r s t r u c t u r e s can h e l p c l a r i f y r e l a t i o n s h i p s . W h i l e more d i s t a n t r e l a t i o n s h i p s a r e b e s t d e t e c t e d by comparison o f p r o t e i n s t r u c t u r e s , i n t e r e s t i n g r e l a t i o n s h i p s may be s e e n by a n a l y s i s o f u n t r a n s l a t e d r e g i o n s o f mRNA s e q u e n c e s . Comparison o f the Schistocerca nitans AKH I and I I cDNA sequences r e v e a l s a h i g h degree o f homology o v e r the t r a n s l a t e d r e g i o n (63.8% i d e n t i t y over 218 n u c l e o t i d e s w i t h 5 g a p s ) , but no s i g n i f i c a n t homology i n the u n t r a n s l a t e d r e g i o n s . T h i s i s i n c o n t r a s t t o the r e l a t i o n s h i p b e t w e e n AKH I cDNA s e q u e n c e s o f S. n i t a n s and S. g r e g a r i a w h i c h show a h i g h d e g r e e o f h o m o l o g y t h r o u g h o u t t h e


231


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available sequences, both translated and untranslated regions (95.2% identity over 330 nucleotides with two gaps). These results strongly suggest that AKH I and II diverged prior to the separation of S. nitans and S. gregaria. Comparison of the Locusta and Schistocerca AKH I precursor carboxyl terminal amino acid sequences reveals an odd result. It happens that there is only one difference between L. nigratoria and S. gregaria while there are two differences between S. gregaria and S. nitans. This might call into question the categorization of these species, but comparison at the cDNA nucleotide level supports the very close relationship between nitans and gregaria. Taghert et al. (22) have illustrated another use of structural comparison by studying the gene structure of the FMRFamide related precursor in both Drosophila melanogaster and Drosophila virilis. They observe interesting short regions of sequence homology 5'to the transcribed region which may indicate conserved regulatory sequences. Thus the application of recombinant DNA technology to the study of neuropeptide biosynthesis has enhanced, and will continue to enhance, our understanding of the structure, function, and evolution of these interesting modulatory agents. Some of the most exciting advances, such as the insights provided by genetic studies, are yet to come. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Nambu, J.R.; Murphy-Erdos, C.; Andrews, P.C.; Feistner, G.J.;Scheller, R.H. Neuron 1988, 1, 55-61. Schneider, L.E.;Taghert, P.H. Proc. Nat. Acad. Sci. USA 1988, 85, 1993-1997. Hekimi, S.; Burkart, W.; Moyer, M.; Fowler, E.;O'Shea, M. Neuron 1989, 2, 1363-1368. Schulz-Allen, M-F.; Roulet, E . ; Fischer-Lougheed, J.;O'Shea, M. Neuron 1989, 2, 1369-1373. Noyes, B.E.;Schaffer, M.H. J. Biol. Chem. 1990, 265, 483489. Chirgwin, J.M.; Przybyla, A.E.; MacDonald, R.J.;Rutter, W.J. Biochemistry 1979, 18, 5294-5299. Schaffer, M.H.; Agarwal, K.L.;Noyes, B.E. Peptides 1982, 3, 693-696. Ilaria, R.; Wines, D.; Pardue, S.; Jamison, S.; Ojeda, S.; Snider, J.;Morrison, M. J . Neurosci. Methods 1985, 15, 165174. Zuker, M.; Stiegler, P. Nucleic Acids Res. 1981, 9, 133-148. Gubler, U.; Chua, A.O.; Hoffman, B.J.; Collier, K.J.; Eng, J . Proc. Natl. Acad. Sci. USA 1984, 81, 4307-4310. Saiki, R.K.; Gelfand, D.H.; Stoffel, S.; Scharf, S . J . ; Higuchi, R.; Horn, G.T.; Mullis, K.B.; Erlich, H.A. Science 1988, 239, 487-491. Nichols, R.; Schneuwly, S.A.;Dixon, J.A. J. Biol. Chem. 1988, 263, 12167-12170. Rees, H.; Shaw, D.D.;Wilkinson, P. Proc. R. Soc. Lond. B 1978, 202, 517-525.





14. Bradfield, J.Y.;Keeley, L . L . J . Biol. Chem. 1989, 264, 12791-12793. 15. Schaffer, M.H., and Noyes, B.E. In Molecular Entomology; Law, J.H., Ed.; Alan R. Liss, New York, 1987; p. 141. 16. Nachman, R.J.; Holman, G.M.; Haddon, W.F.;Ling, N. Science 1986, 234, 71-73. 17. Lechman, R.M.; Wu, P.; Jackson, I.M.D.;Wolf, H. Science 1986, 231, 159-162. 18. von Heijne, G. Nucleic Acids Res. 1986, 14, 4683-4690. 19. Hietter, H.; Luu, B.; Goltzene, F.; Zachary, D.; Hoffmann, J.;Van Dorsselaer, A. Eur. J. Biochem. 1989, 182, 77-84. 20. Comb, M.; Hyman, S.E.;Goodman, H.M. Trends Neurosci. 1987, 10, 473-478. 21. Ziegler, R.; Eckart, K.; Schwarz, H.;Keller, R. Biochem. Biophys. Res. Comm. 1985, 133, 337-342. 22. Taghert, P.; Schneider, L.;O'Brien, M. Internat. Symp. Molec. Insect Sci. 1989, p. 108. RECEIVED September 7, 1990


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