Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.     

The Drosophila ACE3 chorion element autonomously induces amplification.

The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

Localization of a gene for a minor chorion protein in Drosophila melanogaster: a new chorion structural locus

A minor chorion protein (called s70) with an approximate molecular weight of 70,000 D has been characterized in Drosophila melanogaster. The Staket geographic strain was found to carry an electrophoretic variant of this eggshell component and was used to determine the chromosomal location of the s70 gene. Our results establish a new locus for a chorion gene near yellow on the X chromosome and represent the first mapping of a quantitatively minor eggshell protein.     

Nonuniform evolution of duplicated,developmentally controlled chorion genes in a silkmoth

Summary We report the sequence of A/B.L1, a tightly linked pair of genes from the A and B chorion families inBombyx mori. Comparison with the previously characterized A/B.L11 and A/B.L12 pairs from the same species reveals moderate sequence divergence, which is clearly nonuniform. Although the average divergence of A/B.L12 from the other two pairs is more than double that between A/B.L11 and A/B.L1, the ratio differs by more than 30-fold in different DNA regions. One domain of the A gene is highly divergent between A/B.L12 and A/B.L1 or A/B.L11, but essentially invariable in the latter two. In well-aligned DNA segments, nearly all mutated sites (111/112) show variants shared by two of the three sequences, in 42% of the cases between the more distantly related pairs (A/B.L12 and either A/B.L1 or A/B.L11). Eight of the variants shared by distantly related pairs are clustered within 51 bp, suggesting the possibility that they arose through sequence transfers between gene pairs, rather than being primitive or resulting from independent mutations. The short intergenic, putatively regulatory DNa of the developmentally middle A/B.L1 and A/B.L11 pairs resembles that of the late HcA/HcB pairs, particularly in patches that may correspond tocis-regulatory elements.     

NKD, a developmentally regulated tachykinin receptor in Drosophila.

A number of neuropeptides have been described which are present in the insect nervous system. The physiological role of these neuropeptides has not yet been clarified. We have characterized a Drosophila melanogaster cDNA coding for a protein, NKD, whose sequence resembles that of mammalian G protein-coupled neuropeptide receptors. This protein shows 38% homology with the mammalian tachykinin NK3 receptor within the transmembrane domain region. Stable cell lines expressing this cDNA are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. The expression of this gene is detected principally in adult fly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila embryonic development (3 h) and reaches the highest level of expression at 12-16 h, a time which correlates with the period of major neuronal development. In situ hybridization experiments demonstrate that NKD is expressed in the central nervous system, as well as in subsets of neurons in each segment of the developing ventral ganglia. The cytological localization of this gene is at position 86C on the Drosophila third chromosome.     

Seed-specific,developmentally regulated genes of peanut

Four cDNAs of seed-specific and developmentally regulated peanut (Arachis hypogaea L.) genes were identified by differential screening of a peanut-seed cDNA library using cDNA probes constructed from mRNAs isolated from immature and mature stages of the seed. Northern analysis, probed with the four cloned cDNAs, indicated that the genes represented by two cDNAs were expressed abundantly early in seed development, while another two were abundantly expressed later at the cell-expansion stages of seed development. These four genes did not show expression in roots, pegs or leaves. However, one of the early expressed genes was seed coat-specific. One of the clones, Psc11, had significant sequence similarity to subtilisin-like genes in Arabidopsis and soybean. Clones Psc32 and Psc33 had significant similarity to the peanut allergen genes Ara h II and Ara h 6, respectively. The sequence of clone Psc12 was unique and did not show significant similarity to any sequence in the databases. One of the four seed-specific clones showed restriction fragment length polymorphism (RFLP) among peanut lines representing the four peanut botanical varieties. These findings indicate that polymorphism exists in peanut seed-storage genes. This contrasts with other genes previously used for genetic mapping of cultivated peanut. Received: 1 September 2000 / Accepted: 4 May 2001     

DNA sequence of a 3.8 kilobase pair region controlling Drosophila chorion gene amplification

During Drosophila oogenesis, two clusters of chorion genes and their flanking DNA sequences undergo amplification in the ovarian follicle cells. Amplification results from repeated rounds of initiation and bidirectional replication within the chorion gene regions, possibly from a single origin, producing nested replication forks. Previously we have shown that following reintroduction into the Drosophila genome, a specific 3.8 kilobase pair DNA segment from the amplified third chromosome domain could induce developmentally regulated amplification at its site of insertion. Here we present the complete nucleotide sequence of this amplification control element and of genes encoding the chorion structural proteins s18-1 and s15-1, which are contained within it. Sequences that may be involved in the regulation of chorion gene amplification and expression are identified.     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

Acetylcholine is a major excitatory neurotransmitter in the central nervous system of insects. Using DNA probes of the Torpedo nicotinic acetylcholine receptor (AChR) we have isolated two overlapping cDNA clones encoding a putative neuronal AChR protein from the fruitfly, Drosophila melanogaster. The predicted mature protein consists of 497 amino acids, has a calculated mol. wt of 57 340 and shows extensive homology to known AChR subunits from different species along its entire amino acid sequence. Northern analysis revealed a hybridizing mRNA of 3.2 kb in late embryo and in pupae. Expression of the corresponding AChR gene thus characterizes periods of neuronal differentiation in Drosophila.     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

[This corrects the article on p. 1503 in vol. 5.].